GSM8919772: Sorted Memory B and T cells from Patient 9 week 36 – Gene Expression; Homo sapiens; RNA-Seq
At week 0, 9 and 36, PBMCs were isolated from fresh heparin-anticoagulated blood using Ficoll-Paque PLUS gradient (GE Healthcare), washed and incubated with fluorescent antibodies against CD3, CD19, CD45RA, CCR7 and CD27. In case more than one patient sample was analysed at the same day, TotalSeq™-C anti-human Hashtag antibodies (BioLegend) were used. Using a BD AriaII™ cell sorter, we sorted both CD19+CD27+ memory B cells/plasmablasts and CD3+ non-naïve (i.e. not positive for both CCR7 and CD45RA) T cells, and adjusted cell numbers to sequence 10.000 cells per patient and time point. Due to organisational/technical problems, data from patients #6 and #8, as well as the week 36 time point of patient #4 had to be excluded. Single-cell RNA library construction and sequencing was done as previously described.5,55 Briefly, Chromium Next GEM Single Cell 5' reagent kits v2 (dual index) with feature barcode technology for cell surface protein (CITE) mapping (10X Genomics) were used according to the manufacturer's protocol. Final CITE-Seq libraries were generated after index PCR with dual Index Kit TN Set A (10X Genomics) while final GEX and TCR/BCR libraries were generated after fragmentation, adapter ligation and final index PCR with a dual Index Kit TT Set A (10X Genomics). Libraries were quantified using a Qubit HS DNA assay kit (Life Technologies) and fragment sizes were determined using a HS NGS Fragment (1-6000 bp) kit (Agilent). All libraries were sequenced on a NextSeq2000 sequencer (Illumina). GEX and CITE libraries were sequenced using a P3 reagent cartridge (100 cycles) (Illumina) with the following recommended sequencing conditions: read1: 26 nt, index1: 10 nt, read2: 90 nt, index2: 10 nt. The BCR and TCR libraries spiked-in with 10% 2nM PhiX were sequenced using a P3 reagent cartridge (300 cycles) (Illumina) with the following recommended sequencing conditions: read1: 151 nt, index1: 10 nt, read2: 151 nt, index2: 10 nt. The sequencing was performed on a NextSeq2000 (or NextSeq500) device (Illumina) using NextSeq 1000/2000 P3 reagent kits (200 Cycles, Illumina) with the recommended sequencing conditions for 5' GEX and Cite-Seq libraries (read1: 26nt, read2: 90nt, index1: 10nt, index2: 10nt) and NextSeq 1000/2000 P3 reagent kits.