GSM7612597
Single cell suspensions were first incubated with 2.4G2 followed by a biotinylated H-2Kd (SF1-1.1) antibody at room temperature in MACS buffer. The biotinylated CD40LB cells were then removed by incubation with anti-biotin microbeads (Militenyi) and passed through a LS column. The purified B-cells were washed twice with ice-cold PBS and pellets were frozen on dry ice. RNA was isolated from the purified B cells using the RNeasy Mini Kit (Qiagen) and its quality was assessed on a 2100 Bioanalyser (Agilent). Libraries were generated from 100ng of total RNA using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (#E7760) and NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (#E6440) following manufacturer's instructions. cDNA quality was checked using Qubit dsDNA HS Assay Kit (Q32854) and Agilent DNA 5000 tapestation reagents (5067-5588; 5067-5589). Samples were pooled at equimolar quantities, and sequenced on a Novaseq 6000 using one lane of an S1 flowcell, as paired end 50bp (PE50) reads.