GSM7312215: D28_NK_EHMT1_gRNA02_rep01 [bulk RNA-seq]; Homo sapiens; RNA-Seq
DAPI-CD56+CD3- NK cells were isolated by FACS on Day 28 of in vitro differentiation and at least 5,000 cells/sample were  lysed with 2X TCL lysis buffer (Qiagen) to extract total RNA. At least two technical replicates were prepared per sample using the SMART-Seq2 protocol (Picelli et al. 2014), with some modifications. Briefly, RNA was purified from cellular lysate using 2.2X RNA SPRI beads (Beckman Coulter cat. no. A63987) then immediately reverse transcribed in the presence of a template switching oligo (Exiqon) with Maxima RNase H-minus RT (Thermo Fisher Scientific cat. no. EP0751) using a polyT primer containing the ISPCR sequence. Whole transcriptome amplification proceeded with KAPA HiFi HotStart ReadyMix using an ISPCR primer according to the following thermal program: 98C for 3 minutes, 21 cycles of 98C for 15 seconds, 67C for 20 seconds, and 72C for 6 minutes, and a final extension step of 72C for 5 minutes. The amplified cDNA was cleaned with 0.8X DNA SPRI beads (Beckman Coulter cat. no. B23318). Ten nanograms of amplified cDNA was tagmented at 58C for 10 minutes in a 10 uL reaction containing 2 uL of 5X tagmentation buffer (50 mM Tris-HCl, 25 mM MgCl2 pH 8.0), 2 uL of Tris Buffer (10 mM Tris-HCl, 1% Tween-20 pH 8), and 4 uL Nextera Tn5 transposase (Illumina). The reaction was stopped with 1% SDS and incubated at 72C for 10 minutes, then 4C for 3 minutes. The tagmented library was cleaned with 1X DNA SPRI beads followed by an indexing PCR with NEBNext High Fidelity polymerase to incorporate sample index barcodes and Illumina flow cell handles. The index PCR proceeded with the following thermal program: 72C for 3 minutes, 98C for 30 seconds, 12 cycles of 98C for 10 seconds, 60C for 30 seconds, 72C for 30 seconds, and a final extension step of 72C for 5 minutes. The final libraries were pooled, diluted and sequenced on a MiniSeq with a 75-cycle High Output Kit with the following read configuration: 36-8-8-38.