GSM5516678: PBMC sample from subject MuSK-3 at 1 month since first sample collection; Homo sapiens; RNA-Seq
RNA was isolated from frozen peripheral blood mononuclear cells using the RNAeasy Mini kit (Qiagen) per manufacturer's instructions. Bulk libraries were prepared from RNA using reagents from New England Biolabs as part of the NEBNext® Immune Sequencing Kit. cDNA was reverse-transcribed by a template-switch reaction to add a 17-nucleotide unique molecular identifier (UMI) to the 5' end with streptavidin magnetic bead purification. This was then followed by two rounds of PCR; the first round enriched for immunoglobulin sequences using IGHA, IGHD, IGHE, IGHG, and IGHM-specific 3' primers and added a 5' index primer. Libraries were purified with AMPure XP beads (Beckman) after which another round of PCR added Illumina P5 Adaptor sequences to each amplicon. The number of PCR cycles were selected based on real-time quantitative PCR to avoid the plateau phase. Libraries were then purified again with AMPure  XP beads. Libraries were pooled in equimolar libraries and sequenced by 325 cycles for read1 and 275 cycles for read 2 using paired-end sequencing with a 20% PhiX spike on the Illumina MiSeq platform according to manufacturer's recommendations.