Smart-Seq2 for Bulk RNA-Seq
Population RNA-seq was performed as described (Ordovas-Montanes et al., 2018, Trombetta et al., 2014). Briefly, RNA from population lysates was purified using AMPure RNA Clean Spri beads (Beckman Coulter) at a 2.2x volume ratio, and mixed with oligo-dT primer, dNTPs (NEB), and RNase inhibitor (Fisher Scientific) at 72°C for 3 min on a thermal cycler to anneal the 3′ primer to polyadenylated mRNA. Reverse transcription was carried out in a master mix of Maxima RNaseH-minus RT enzyme and buffer (Fisher Scientific), MgCl2 (Sigma), Betaine (Sigma), RNase inhibitor, and a 5′ template switch oligonucleotide, and PCR was carried out using KAPA HiFi HotStart ReadyMix (Kapa Biosystems) and IS PCR primer and amplified for 18 cycles. Libraries were purified using AMPure XP SPRI beads at a volume ratio of 0.8x followed by 0.9x. Library size was assessed using a High-Sensitivity DNA chip (Agilent Bioanalyzer), confirming the expected size distribution of ∼1,000-2,000 bp. Tagmentation reactions were carried out with the Nextera XT DNA Sample Preparation Kit (Illumina) using 250 pg of cDNA per single cell as input, with modified manufacturer’s instructions as described. Libraries were purified twice with AMPure XP SPRI beads at a volume ratio of 0.9x, size distribution assessed using a High Sensitivity DNA chip (Agilent Bioanalyzer) and Qubit High-Sensitivity DNA kit (Invitrogen). Libraries were pooled and sequenced using NextSeq500/550 High Output v2 kits (75 cycles, Illumina) using 30-30 paired end sequencing with 8-mer dual indexing.