title: GSM3030485: Blimp-1-GFPhighCD138+PBS_1; Mus musculus; RNA-Seq
library_construction_protocol: Cells were sorted directly into 8µl lysis mix as described in library construction protocol. RNA libraries to sequence 40-50 cells were prepared using an adapted Smart-seq2 protocol (Picelli et al., Full-length RNA-seq from single cells using Smart-seq2. Nat Protocols 2014). Cells were sorted directly into 8µl lysis mix. Reaction volumes was doubled relative to Picelli et al. from reverse transcription to preamplification in order to help with facs stream alignment into 0.2ml PCR capture tubes. Normal volumes were used at all post pre-amplification steps. 17 or 18 cycles of PCR amplification were used for GC and follicular samples, respectively. cDNA purification was performed usign Ampure XP magnetic beads at a sample:bead ratio of 1.7 because this was found to reduce primer-dimer contamination. Libraries were analysed for size and quality with a High Sensitivity Analyser (Agilent). cDNA tagmentation was performed with the Nextera XT DNA Sample Preparation kit (Illumina). Libraries were quantified using PicoGreen (Illumina), sized using the High Sensitivity Analyser and equal amounts of tagmented cDNA from each library were pooled. Pools containing concentrations of approximately 10 nM were sequenced on a NextSeq500 using FC-404-2005 NextSeq 500/550 High Output Kits v2 (75 cycles).