{"id":377,"date":"2024-04-25T20:04:00","date_gmt":"2024-04-25T20:04:00","guid":{"rendered":"https:\/\/winterdev.cc\/nwgc-dev\/?page_id=377"},"modified":"2024-04-25T20:04:00","modified_gmt":"2024-04-25T20:04:00","slug":"sample-requirements","status":"publish","type":"page","link":"https:\/\/nwgc.gs.washington.edu\/wordpress\/services\/functional-genomics\/fiberseq\/sample-requirements\/","title":{"rendered":"Submitting Samples"},"content":{"rendered":"<p><strong>***All sample drop-offs must be scheduled ahead of time due to the sensitivity of the 10X Protocol.<\/strong>***<\/p>\n<p>Cryopreserved PBMCs or tissues are to be kept at -80C or on dry ice, to preserve sample integrity, from the time of collection to the time of delivery. <\/p>\n<p>The input material for the single-cell multiome ATAC + Gene expression workflow is a clean nuclei suspension. This nuclei suspension may come from tissue or a cell suspension. Whenever possible, dissociate tissues into a cell suspension before isolating nuclei. However, for frozen and hard to dissociate tissues it is necessary to go straight from tissue to nuclei. If you start with a low viability cell suspension, sorting for live cells and\/or removing dead cells, with the dead cell removal kit, before nuclei isolation may improve sample quality. After nuclei isolation, stain cells with the viability dye to determine quality. Less than 5% of cells should be alive after nuclei isolation. Sorting after nuclei isolation may be necessary to remove the debris.<\/p>\n<p><strong>Minimum cell\/nuclei sample requirements:<\/strong><\/p>\n<p>The optimal input nuclei concentration for most 10x Genomics Single Cell assays is 700\u20131,200 nuclei\/\u03bcl. <\/p>\n<p class=\"has-text-align-left\">*If possible, bring the input nuclei suspension to a concentration that is optimal for the dynamic range of counting technique used (manual or automated), allows for 2\u20133 reproducible counts (where the standard<br \/>deviation is &lt;25%), and requires pipetting 2\u20135 \u03bcl (Single Cell Multiome ATAC + Gene Expression and Single Cell ATAC Assays) into the Single Cell Master Mix.<\/p>\n<p>*Nuclei recovery from healthy tissues typically ranges from 5,000\u201315,000 nuclei\/mg. Both disease state  and cell type composition can impact nuclei recovery. <\/p>\n<p>*Resuspend nuclei in a low initial volume (50\u2013200 \u03bcl) during final resuspension step if nuclei yield is expected to be low or is unknown.<\/p>\n<p>*DO NOT resuspend nuclei in a volume &lt;50 \u03bcl regardless of input tissue<br \/>mass<\/p><\/p>\n","protected":false},"excerpt":{"rendered":"<p>***All sample drop-offs must be scheduled ahead of time due to the sensitivity of the 10X Protocol.*** Cryopreserved PBMCs or tissues are to be kept at -80C or on dry ice, to preserve sample integrity, from the time of collection to the time of delivery. The input material for the single-cell multiome ATAC + Gene expression workflow is a clean nuclei suspension. This nuclei suspension may come from tissue or a cell suspension. Whenever possible, dissociate tissues into a cell&#8230;<\/p>\n<div><a class=\"more-link\" href=\"https:\/\/nwgc.gs.washington.edu\/wordpress\/services\/functional-genomics\/fiberseq\/sample-requirements\/\">Continue reading <span class=\"screen-reader-text\">Submitting Samples<\/span><\/a><\/div>\n","protected":false},"author":2,"featured_media":0,"parent":320,"menu_order":2,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"class_list":["post-377","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/nwgc.gs.washington.edu\/wordpress\/wp-json\/wp\/v2\/pages\/377","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/nwgc.gs.washington.edu\/wordpress\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/nwgc.gs.washington.edu\/wordpress\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/nwgc.gs.washington.edu\/wordpress\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/nwgc.gs.washington.edu\/wordpress\/wp-json\/wp\/v2\/comments?post=377"}],"version-history":[{"count":0,"href":"https:\/\/nwgc.gs.washington.edu\/wordpress\/wp-json\/wp\/v2\/pages\/377\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/nwgc.gs.washington.edu\/wordpress\/wp-json\/wp\/v2\/pages\/320"}],"wp:attachment":[{"href":"https:\/\/nwgc.gs.washington.edu\/wordpress\/wp-json\/wp\/v2\/media?parent=377"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}