GSM5861295: RA134 Peripheral Blood; Homo sapiens; RNA-Seq
Cryopreserved synovial fragments were thawed. Synovial tissue fragments were then disaggregated by combination of an enzymatic digestion and mechanical disruption. PBMCs were thawed by rapidly warming the cryovial in a 37 °C water bath. To stain cells for sorting, cells were first incubated with Fc-Block (Biolegend) for 5 minutes at room temperature. Cells were then incubated with a mixture of fluorochrome-conjugated antibodies for 30 minutes on ice, washed with PBS and then resuspended in PBS solution containing DAPI (Biolegend). Cells were sorted on a FACSAria II (BD biosciences) into 5%FBS/RPMI media using a 100-μm nozzle. B cells were gated as CD45+CD3-CD14-CD19+. Cellular suspensions were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA). Single-cell RNA-Seq libraries were prepared using Chromium Single-Cell 5′ Library & Gel Bead Kit (PN-1000006, PN-1000014, 10x Genomics).  Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by End Repair, A-tailing, Adaptor Ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification. Paired end reads of 150nt were generated for each B-cell enriched library using the MiSeq sequencer (Illumina). The 5' gene expression libraries were sequenced following 10X Genomics read length guidelines on the NovaSeq6000 sequencer (Illumina).