GSM7790881: Replicate 11, dialout library; Homo sapiens; OTHER
Nuclei were prepared according to 10X Genomics' Nuclear Isolation for Single Cell ATAC Sequencing protocol with the following notes. IGEPAL CA-630 was used for the lysis buffer a 10% solution of which was prepared fresh on the day of nuclear isolation using 100% stock (Sigma i8896). After trypsinization and pelleting, cells were washed twice with ice cold PBS + 0.04% BSA before following the remainder of the nuclear isolation protocol. Lysis occurred for 4 minutes which previously was found to be appropriate time for cytoplasmic removal while retaining nuclear membrane architecture by DIC microscopy. Cells were resuspended in diluted nuclei buffer and triplicates were counted before proceeding with Spear-ATAC sequencing as previously published. Library construction was performed according to 10X Genomics scATAC-seq v2 Chemistry and Spear-ATAC with the following modifications and notes. 10,000 nuclei were targeted for capture per 10X chip lane. Spear-ATAC included the following changes to the 10X scATAC library preparation protocol: the 1.2 uL per reaction of 50 uM oSP1735 oligonucleotide is spiked into the GEM generation master mix and the in-GEM PCR was increased from 12 to 15 cycles. Single-cell ATAC library fragment size distribution was analyzed by Tapestation prior to preparing enriched dial-out libraries for perturbation genotyping. The dial-out libraries were again prepared according to the Spear-ATAC protocol with the following changes: linear PCR with biotinylated oSP2053 was increased to 30 total cycles and the exponential PCR with oSP1594 and indexed P7 primers was increased to 18 total cycles. The indices on the P7 primers were increased from 8 bases to 10 bases.