GSM8832706: Patient 6; Homo sapiens; RNA-Seq
Frozen tissue (0.5-2.0 cm) into 1.0 mL cold 292 mM NaCl 29.2, 2 mM CaCl2, 42 mM MgCl2, 20 mM Tris-HCl pH 8.0, 1% CHAPs, 2% BSA, in Ultrapure H2O (CST buffer) into one well of a 6-well plate. Tissue was minced in CST buffer with spring scissors for 10 minutes on ice until homogenized. The cell/tissue solution was through a 40 µm cell strainer. The well was washed with 1.0 ml CST and filtered. Samples were filtered into a 15 mL canonical tube using 5.0 ml CST lacking CHAPs and BSA (ST buffer). Filtered samples were centrifuged at 4oC for 5 min at 500×g. The supernatant was removed, and the pellet resuspended in 1% BSA with RNAsin Plus in phosphate buffered saline (Nuclei Suspension Buffer). The nuclei were suspended in up to 500 µl nuclei suspension buffer with approximately 1200 nuclei per µl. Nuclei were filtered again through a 35 µm cell strainer (FACS filter cap tube). Nuclei were counted using a hemocytometer. An aliquot was subsequently resuspend in a 1:1000 dilution of Hoescht and TOPRO-3 for visualization of nuclei integrity as we have previously described85. Nuclei were transported on ice until gel-bead-in-emulsion (GEM) generation. Libraries were made after GEM generation in accordance with 10X Genomics Single Cell 3' kit v3.1 (10x Genomics, #1000269) according to the manufacturer's guidelines and Chromium Next GEM Single Cell 3' v. 3.1 (Dual Index Kit TT Set A, 10X Genomics, #1000215) protocol. The libraries were quantified and qualified using Qubit 4 Fluorometer (Thermo Fisher Scientific, #Q33238, 1X dsDNA, high sensitivity, 500 rxns (Thermo Fisher Scientific, #Q33231) and TapeStation 4150 (Aglient, #G2992AA, High Sensitivity D5000 ScreenTape Aglient, #5067-5592, High Sensitivity D5000 Ladder Agilent, #5067-5594. Libraries were constructed using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (New England Biolabs Inc., #E7770S) and the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc., #E7490S). These libraries were dual-indexed using the NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) (New England Biolabs Inc., E7600S). The libraries were quantified and qualified using Qubit (Thermo-Fischer, #Q32852) and TapeStation (Agilent, # 5067-5579). Pooled libraries were sequenced using 150 bp paired-end reads on a NovaSeq 6000 SP v1.5 (300 cycles) flow cell (Illumina, #20028400).