GSM8580159: Donor26_AIM2; Homo sapiens; RNA-Seq
No RNA extraction. Cells directly sorted into low-input lysis buffer (LI-LB) containing 0.1% Triton X-100 (vol/vol), 1U/µl RNase inhibitor (Takara), 2.5mM dNTPs (Thermo Fisher Scientific). Double-stranded (ds)-cDNA synthesis and amplification using the Smart-seq2 protocol. Briefly, poly-dT and template-switch oligos were used for mRNA capture and generation of full-length double-stranded cDNA using SuperScript II reverse transcriptase kit (Thermo Fisher). The cDNA was amplified using KAPA HiFi HotStart ReadyMix buffer and purified with AMPure XP magnetic bead (Beckman Coulter). Quality and quantity of cDNA were assessed with Tapestation (Agilent) and Qubit (Invitrogen). For each sample, 1 ng cDNA was used to prepare dual-index barcoded libraries using Nextera XT DNA library prep kit and index kits (Illumina). Libraries were purified with AMPure XP magnetic bead. Good quality libraries, as examined with Tapestation and Qubit, were retained for sequencing. Samples from low, mid and high range Gensini scores (severity of coronary artery disease) were processed together to minimize batch effects. Equimolar concentrations of uniquely indexed libraries were pooled and sequenced using an S4 flow cell on a NovaSeq 6000 (Illumina) to obtain 70 million 100-bp paired-end reads per cluster.