GSM6467424: Sample 3, mRNA derived cDNA; Homo sapiens; RNA-Seq
Nuclei from fresh frozen tissues were isolated following the Nuclei Pure Prep nuclei isolation kit (Sigma-Aldrich, Catalog ID: NUC201) with some modifications. To summarize, ~10mg of tissue were washed with PBS to remove extraneous OCT the samples were frozen in. The tissue was homogenized with both wide and narrow pestles submerged in 2.5mL of the lysis buffer in a Dounce homogenizer. The lysate mixed with 4.5mL 1.8M sucrose cushion were gently layered on top of the 2.5mL of 1.8M sucrose cushion in Beckman ultracentrifuge tubes. Samples were centrifuged for 45 min at 13,000 RPM at 4°C in an ultracentrifuge. Samples were multiplexed with lipid-tagged oligonucleotides following the MULTI-seq protocol19. Nuclei were resuspended in 1% BSA PBS and filtered with 70um and 40um Flowmi filters. Library was performed according to the manufacter's instructions (single cell 3' protocol, 10x Genomics). Cellular mRNA and hashtag oligonucleotides were reverse-transcribed and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR according to the Chromium Single Cell 3' protocol (10x Genomics) and with specific primers amplifying the HTO-derived tags.