{"id":2780,"date":"2020-06-02T14:49:31","date_gmt":"2020-06-02T18:49:31","guid":{"rendered":"https:\/\/www.med.unc.edu\/genomics\/?page_id=2780"},"modified":"2023-02-16T19:17:39","modified_gmt":"2023-02-17T00:17:39","slug":"submission-requirements","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/genomics\/submissions\/submission-requirements\/","title":{"rendered":"Sample Preparation Requirements for Submission"},"content":{"rendered":"<p>&nbsp;<\/p>\n<span class=\"collapseomatic \" id=\"id69d41553046aa\"  tabindex=\"0\" title=\"RNA and DNA Extractions\"    >RNA and DNA Extractions<\/span><div id=\"target-id69d41553046aa\" class=\"collapseomatic_content \">\n<p>The HTSF does not specialize in DNA and RNA extractions; therefore, we recommend that you speak with fellow researchers in your field or please <a href=\"https:\/\/bsp.web.unc.edu\/\">visit the BioSpecimen Processing Facility (BSP).<\/a> The BSP can assist with RNA and DNA extractions and offer recommendations.<\/p>\n<p>The DNA or RNA extraction method can be critical to successful creation of your sequencing library. In our experience, most commercial kits that use a column purification step can be used.<\/p>\n<ul>\n<li>If you use Trizol or phenol\/chloroform to extract RNA or DNA, you must perform a clean-up step using a column based clean-up kit before submitting to HTSF.<\/li>\n<li>HTSF does not recommend resin based nucleic acid purification.<\/li>\n<li>Do not use glycogen as a carrier during ethanol precipitation of RNA or DNA samples. Instead, we recommend using linear polyacrylamide (LPA) which is available from Sigma-Aldrich (Cat #56575), Fisher Scientific (J67830-XF), and other vendors.<\/li>\n<\/ul>\n<h3>RNA Purification<\/h3>\n<p><span style=\"background-color: #ffff00\">Many of our users have found the following kits work well for their purposes. HTSF cannot confirm they will work for your specific needs. For example, when doing long read technology, it is critical to preserve the material size. The guidelines in the tables below for recommended purification methods based on the type of material being submitted.<\/span><\/p>\n<table style=\"height: 162px;width: 100%;border-collapse: collapse;border-style: solid;border-color: #007FAE\" border=\"3\">\n<tbody>\n<tr style=\"height: 23px\">\n<td style=\"width: 49.9398%;height: 23px\" width=\"234\"><strong style=\"color: #007fae\">Type of Material<\/strong><\/td>\n<td style=\"width: 49.9398%;height: 23px\" width=\"234\"><strong style=\"color: #007fae\">Purification Method<\/strong><\/td>\n<\/tr>\n<tr style=\"height: 53px\">\n<td style=\"width: 49.9398%;height: 53px\" width=\"234\"><strong>Total RNA<\/strong><\/td>\n<td style=\"width: 49.9398%;height: 53px\" width=\"234\">\n<ul>\n<li>Zymo Research Quick RNA Plus Kits (variable capacities)<\/li>\n<\/ul>\n<\/td>\n<\/tr>\n<tr style=\"height: 53px\">\n<td style=\"width: 49.9398%;height: 53px\" width=\"234\"><strong>RNA from FFPE<\/strong><\/td>\n<td style=\"width: 49.9398%;height: 53px\" width=\"234\">\n<ul>\n<li>MagMax\u2122 Total Nucleic Acid Isolation Kit<\/li>\n<li>RecoverAll\u2122 Total Nucleic Acid Isolation Kit for FFPE<\/li>\n<\/ul>\n<\/td>\n<\/tr>\n<tr style=\"height: 33px\">\n<td style=\"width: 49.9398%;height: 33px\" width=\"234\"><strong>For smallRNA (enrichment)<\/strong><\/td>\n<td style=\"width: 49.9398%;height: 33px\" width=\"234\">\n<ul>\n<li>PureLink\u2122 miRNA Isolation Kit<\/li>\n<\/ul>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<h3>DNA Purification<\/h3>\n<table style=\"width: 100%;border-collapse: collapse;border-style: solid;border-color: #007FAE\" border=\"3\">\n<tbody>\n<tr>\n<td style=\"width: 49.9398%\" width=\"234\"><strong style=\"color: #007fae\">Type of Material<\/strong><\/td>\n<td style=\"width: 49.9398%\" width=\"234\"><strong style=\"color: #007fae\">Purification Method<\/strong><\/td>\n<\/tr>\n<tr>\n<td style=\"width: 49.9398%\" width=\"234\"><strong>DNA<\/strong><\/td>\n<td style=\"width: 49.9398%\" width=\"234\">\n<ul>\n<li>Zymo Research Quick DNA Plus Kits (variable capacities)<\/li>\n<\/ul>\n<\/td>\n<\/tr>\n<tr>\n<td style=\"width: 49.9398%\" width=\"234\"><strong>High Molecular Weight DNA<\/strong><\/p>\n<p><strong>(Oxford Nanopore sequencing)<\/strong><\/td>\n<td style=\"width: 49.9398%\" width=\"234\">\n<ul>\n<li>Beckman Coulter DNAdvance Kit (from tissue)<\/li>\n<li>Beckman Coulter GenFind V3 Kit (from blood)<\/li>\n<li>Circulomics Nanobind CBB Big DNA Kit (cells, bacteria, blood)<\/li>\n<li>Circulomics Nanobind Tissue Big DNA Kit<\/li>\n<\/ul>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n<span class=\"collapseomatic \" id=\"id69d4155304810\"  tabindex=\"0\" title=\"Sample Quantification\"    >Sample Quantification<\/span><div id=\"target-id69d4155304810\" class=\"collapseomatic_content \"><img loading=\"lazy\" decoding=\"async\" class=\"alignleft\" src=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/08\/working_qubit1-scaled.jpg\" alt=\"qubit\" width=\"131\" height=\"183\" \/><\/p>\n<p>We recommend using a spectrophotometer(Nanodrop) to check the purity of your samples before submitting to HTSF. HTSF performs rigorous QAQC analysis to assess nucleic acid quality and concentration; however, we do not use a spectrophotometer. During the submission process, there will be an option to receive QAQC updates and\/or approve QAQC results as samples move through the queue.<\/p>\n<\/div>\n<span class=\"collapseomatic \" id=\"id69d41553048ca\"  tabindex=\"0\" title=\"Sample Diluent Requirements\"    >Sample Diluent Requirements<\/span><div id=\"target-id69d41553048ca\" class=\"collapseomatic_content \">\n<p>Samples may be submitted in one of the following diluents:<img loading=\"lazy\" decoding=\"async\" class=\"alignright\" src=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/08\/artistic_solutions_blk.jpg\" alt=\"artistic solutions\" width=\"200\" height=\"823\" \/><\/p>\n<ul>\n<li>dH2O (preferred)<\/li>\n<li>EB buffer (10 mM Tris-Cl, pH 8.5)<\/li>\n<li>RSB buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2)<\/li>\n<\/ul>\n<p><strong>TE buffer is NOT acceptable and samples in this buffer will not be accepted or will incur an additional charge to correct this problem.<\/strong><\/p>\n<p>EDTA in these buffers interferes with downstream processing and can prevent the sequencing process from working correctly. Samples in TE buffer need to be cleaned up and re-suspended in an appropriate solution\u00a0<strong>PRIOR<\/strong>\u00a0to submission.<\/p>\n<\/div>\n<span class=\"collapseomatic \" id=\"id69d41553049a9\"  tabindex=\"0\" title=\"Sample Requirements\"    >Sample Requirements<\/span><div id=\"target-id69d41553049a9\" class=\"collapseomatic_content \">\n<p>Below are general tables to follow when submitting material to the HTSF. These tables refer to the volume and concentration requirements depending on the submitted material type and preparation method. See the <a href=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/07\/HTSF-DNA-and-RNA-Sample-Preparation-Requirement-Guide_V6_12-2021_FINAL-1.pdf\">HTSF DNA and RNA Sample Preparation Requirement Guide<\/a> on the <em>Forms and Guide page<\/em> under <em>General<\/em> for specifics for your library method of choice.<\/p>\n<h3>DNA Samples<\/h3>\n<table style=\"height: 125px;width: 100%;border-collapse: collapse;border-style: solid;border-color: #007FAE\" border=\"3\">\n<tbody>\n<tr style=\"height: 23px\">\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong style=\"color: #007fae\">Preparation Method<\/strong><\/td>\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong style=\"color: #007fae\">Minimum Required Volume<\/strong><\/td>\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong style=\"color: #007fae\">Minimum Concentration<sup>1<\/sup><\/strong><\/td>\n<\/tr>\n<tr style=\"height: 33px\">\n<td style=\"width: 33.3333%;height: 33px;text-align: center\"><strong>DNAseq<\/strong><\/td>\n<td style=\"width: 33.3333%;height: 33px;text-align: center\">60 \u00b5l<\/td>\n<td style=\"width: 33.3333%;height: 33px;text-align: center\">30-40 ng\/\u00b5l<\/td>\n<\/tr>\n<tr style=\"height: 23px\">\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong>DNA Nextera XT<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\">15 \u00b5l<\/td>\n<td style=\"width: 33.3333%;text-align: center\">1 ng\/\u00b5l<br \/>\n(200 ng total)<\/td>\n<\/tr>\n<tr style=\"height: 23px\">\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong>DNA Sure Select<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\">20-60 \u00b5l<\/td>\n<td style=\"width: 33.3333%;text-align: center\">25 ng\/\u00b5l<br \/>\n(3 \u00b5g total)<\/td>\n<\/tr>\n<tr style=\"height: 23px\">\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong>CHiP DNA<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\">12 \u00b5l<\/td>\n<td style=\"width: 33.3333%;text-align: center\">2 ng\/\u00b5l<sup>2<\/sup><br \/>\n(20 ng total for library preparation)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<h3>RNA Samples<\/h3>\n<table style=\"height: 277px;width: 100%;border-collapse: collapse;border-style: solid;border-color: #007FAE\" border=\"3\">\n<tbody>\n<tr style=\"height: 23px\">\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong style=\"color: #007fae\">Preparation Method<\/strong><\/td>\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong style=\"color: #007fae\">Required Volume<\/strong><\/td>\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong style=\"color: #007fae\">Minimum Concentration<\/strong><\/td>\n<\/tr>\n<tr style=\"height: 33px\">\n<td style=\"width: 33.3333%;height: 33px;text-align: center\" width=\"172\"><strong>Total RNA (TruSeq RiboZero Gold)<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\" rowspan=\"4\" width=\"172\">60 \u00b5l<\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">30-40 ng\/\u00b5l<\/td>\n<\/tr>\n<tr style=\"height: 33px\">\n<td style=\"width: 33.3333%;height: 33px;text-align: center\" width=\"172\"><strong>Total RNA (Roche Seq KAPA)<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">30 ng\/\u00b5l<\/td>\n<\/tr>\n<tr style=\"height: 33px\">\n<td style=\"width: 33.3333%;height: 33px;text-align: center\" width=\"172\"><strong>Total RNA (MicroRNA)<sup>\u00a0<\/sup><\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">67 ng\/\u00b5l<br \/>\n(2 \u00b5g total)<\/td>\n<\/tr>\n<tr style=\"height: 33px\">\n<td style=\"width: 33.3333%;height: 33px;text-align: center\" width=\"172\"><strong>mRNA (KAPA stranded)<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">30 ng\/\u00b5l<\/td>\n<\/tr>\n<tr style=\"height: 33px\">\n<td style=\"width: 33.3333%;height: 33px;text-align: center\" width=\"172\"><strong>mRNA (KAPA non-stranded)<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">25 \u00b5l<\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">10 ng\/\u00b5l<\/td>\n<\/tr>\n<tr style=\"height: 33px\">\n<td style=\"width: 33.3333%;height: 33px;text-align: center\" width=\"172\"><strong>Small RNA (purified)<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">30 \u00b5l<\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">67 ng\/\u00b5l<\/p>\n<p>*works down to 1ng, so submission concentrations can be very low<\/td>\n<\/tr>\n<tr style=\"height: 33px\">\n<td style=\"width: 33.3333%;height: 33px;text-align: center\" width=\"172\"><strong>Amplicons<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">60 \u00b5l<\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">20 \u2013 30 ng\/\u00b5l<\/td>\n<\/tr>\n<tr style=\"height: 23px\">\n<td style=\"width: 33.3333%;height: 23px;text-align: center\"><strong>FFPE RNA<\/strong><\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">15 \u00b5l<\/td>\n<td style=\"width: 33.3333%;text-align: center\" width=\"172\">500-4000 ng<sup>2<\/sup><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>For more detailed sample submission requirements please refer to <a href=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/06\/HTSF-Submission_Sample_Requirements_2020.xlsx\">Submission Sample Requirements.<\/a><\/p>\n<\/div>\n<span class=\"collapseomatic \" id=\"id69d4155304a67\"  tabindex=\"0\" title=\"Library and Pool Submissions\"    >Library and Pool Submissions<\/span><div id=\"target-id69d4155304a67\" class=\"collapseomatic_content \">\n<ul>\n<li>The minimum overall submitted volumes are listed below<\/li>\n<li>If you are submitting a POOL, the combined volume must be at least the minimum volume listed.<\/li>\n<li>If you are submitting for any standard loading version of the NovaSeq, Please reference <a href=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/07\/HTSF-NovaSeq-Requirements-and-Pooling-Suggestions_v3_7-2020_FINAL.pdf\">NovaSeq Requirements Table<\/a> on the <em>Forms and Guides page<\/em> under Illumina. NovaSeqS1, S2, S4 requires larger volumes for loading than the MiSeq platforms. Contact HTSF for other options if you cannot meet these requirements.<\/li>\n<\/ul>\n<table style=\"width: 100%;border-collapse: collapse;border-style: solid;border-color: #007FAE\" border=\"3\">\n<tbody>\n<tr>\n<td style=\"width: 33.4537%;text-align: center\" width=\"104\"><strong style=\"color: #007fae\">Material<\/strong><\/td>\n<td style=\"width: 31.2876%;text-align: center\" width=\"104\"><strong style=\"color: #007fae\">Volume<\/strong><\/td>\n<td style=\"width: 35.2587%;text-align: center\" width=\"104\"><strong style=\"color: #007fae\">Molarity*<\/strong><\/td>\n<\/tr>\n<tr>\n<td style=\"width: 33.4537%;text-align: center\" width=\"104\"><strong>Single library<\/strong><\/td>\n<td style=\"width: 31.2876%;text-align: center\" rowspan=\"2\" width=\"104\">20 uL<\/td>\n<td style=\"width: 35.2587%;text-align: center\" width=\"104\">15 nM (2 nM\u00a0minimum)<\/td>\n<\/tr>\n<tr>\n<td style=\"width: 33.4537%;text-align: center\" width=\"104\"><strong>Project\/study pooled libraries<\/strong><\/td>\n<td style=\"width: 35.2587%;text-align: center\" width=\"104\">15 nM (5nM\u00a0minimum) total pool<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n<span class=\"collapseomatic \" id=\"id69d4155304b20\"  tabindex=\"0\" title=\"Required Sample Containers\"    >Required Sample Containers<\/span><div id=\"target-id69d4155304b20\" class=\"collapseomatic_content \">\n<p>For specific information on the submission container requirements please refer to the <a href=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/07\/HTSF-Approved-Container-Requirements_v5_7-2020_FINAL.pdf\">HTSF Approved Container Requirements<\/a> found on the <em>Forms and Guides page<\/em> under <em>TracSeq and Submissions<\/em>. This guide includes specifics for all approved containers, including vendor info for some containers. Additionally, there are images for all approved and non-approved containers.<\/p>\n<h3>Tubes<\/h3>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignright\" src=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/08\/Containers_snap-cap-tubes.jpg\" alt=\"containers snap-cap tubes\" width=\"150\" height=\"122\" \/>HTSF accepts<\/p>\n<ul>\n<li style=\"list-style-type: none\">\n<ul>\n<li>1.5 &#8211; 1.7 ml polypropylene snapcap or screwcap<\/li>\n<li>0.5 ml Matrix tubes\n<ul>\n<li>A note, submission in Matrix tubes is preferred when more than 48 samples are planned for a submission<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignright\" style=\"padding-right: 16px\" src=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/08\/Containers_matrix.jpg\" alt=\"containers matrix\" width=\"200\" height=\"111\" \/><\/p>\n<ul>\n<li style=\"list-style-type: none\">\n<ul>\n<li>HTSF has high throughput pipelines established for processing matrix tubes<\/li>\n<li>At this time, Matrix tubes are preferred over 96 well plates because of flexibility for processing<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>We\u00a0do not\u00a0accept<\/p>\n<ul>\n<li>Dolphins tubes<\/li>\n<li>any tube &gt;1.7 ml or &lt;0.5 ml.<\/li>\n<\/ul>\n<h3>Plates<\/h3>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignleft\" src=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/08\/Containers_plate.jpg\" alt=\"containers plate\" width=\"180\" height=\"126\" \/>The HTSF can\u00a0<strong>ONLY<\/strong>\u00a0accept BioRad (catalog\u00a0HSP-9631) 96 well Skirted hard-shelled plates. The HTSF can supply limited numbers of these 96 well plates as needed at a small cost. Any other formatted 96 well plate will be returned to the study or fees will be incurred for transfer of samples to new plate. HTSF requires these specifics plates due to our robotics.<\/p>\n<p>For more specific information on the submission container requirements please refer to the <a href=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/07\/HTSF-Approved-Container-Requirements_v5_7-2020_FINAL.pdf\">HTSF Container Information Guide<\/a>, also found on the <em>Forms and Guides page<\/em> under <em>General<\/em>.<\/p>\n<\/div>\n<h2 class=\"section-som\"><span>Relevant Forms and Guides<\/span><\/h2>\n<a href=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/07\/HTSF-DNA-and-RNA-Sample-Preparation-Requirement-Guide_V6_12-2021_FINAL-1.pdf\">HTSF DNA and RNA Sample Preparation Requirement Guide <\/a><br \/>\n<a href=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/07\/HTSF-NovaSeq-Requirements-and-Pooling-Suggestions_v3_7-2020_FINAL.pdf\">NovaSeq Requirements Table <\/a><br \/>\n<a href=\"https:\/\/www.med.unc.edu\/genomics\/wp-content\/uploads\/sites\/708\/2020\/07\/HTSF-Approved-Container-Requirements_v5_7-2020_FINAL.pdf\">HTSF Approved Container Requirements<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>&nbsp; HTSF DNA and RNA Sample Preparation Requirement Guide NovaSeq Requirements Table HTSF Approved Container Requirements<\/p>\n","protected":false},"author":64010,"featured_media":0,"parent":2772,"menu_order":2,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"layout":"","cellInformation":"","apiCallInformation":"","footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2780","page","type-page","status-publish","hentry","odd"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.8 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Sample Preparation Requirements for Submission - High-Throughput Sequencing Facility (HTSF)<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.med.unc.edu\/genomics\/submissions\/submission-requirements\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Sample Preparation Requirements for Submission - High-Throughput Sequencing Facility (HTSF)\" \/>\n<meta property=\"og:description\" content=\"&nbsp; 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