GSM5942456: scRNAseq CD4 T cells from patient X205; Homo sapiens; RNA-Seq
CD45+ cells were sorted into CD45+ fractions that were either CD3- CD19- or positive for either CD3 or CD19 and CD45+ CD3- CD19- populations were sequenced separately from CD3+ and CD19+ populations. Single cells were partitioned using the BD Rhapsody system per the manufacturer's instructions. Libraries were created following the BD Rhapsody™ System VDJ CDR3, Sample Tag, and BD AbSeq Library Preparation Protocol. In more detail: Single-cell libraries were prepared using the BD Rhapsody Express system (BD Biosciences, #633707) and Targeted mRNA and AbSeq Reagent Kit-4 pack (BD Biosciences, #633771) according to the manufacturer's protocol (BD Biosciences). Briefly, tumor samples from each donor were thawed and labelled with sample tags using BD Single-Cell Multiplexing Kit (BD Biosciences, #633781). Cells from each donor were labelled with a unique sample tag. Cells were then washed in BD Stain buffer (BD Biosciences, #554656), pooled together and incubated in Fc block followed by labelling cells with BD AbSeq Ab-Oligos master mix (3uL per Ab-Oligo). For flow sorting various lymphoid and myeloid cell populations, cells were stained with a mixture of fluorescently labelled antibodies (BD Biosciences or BioLegend): anti-human CD3 (#OKT3), CD4 (#RPA-T4), CD8 (#RPA-T8), CD19 (#HIB19), CD45 (#2D1), for 30 mins at 4ºC. After sorting, cells were counted and pooled such that there were 20,000 cells resuspended in 620 uL of BD Sample buffer. The BD Rhapsody cartridges were primed, and the pooled cells were loaded onto the cartridges and incubated at room temperature. Next, Cell Capture beads were washed & loaded onto the cartridge. After incubation, cartridges were washed twice with BD Sample Buffer. This was followed by lysis of cells and retrieval of beads. After washing the retrieved Cell Capture beads, we proceeded immediately with reverse transcription. For the subsequent steps, we switched to BD Rhapsody system VDJ CDR3, Sample Tag, and BD AbSeq library protocol (BD Biosciences) to generate TCR and BCR libraries in addition to mRNA, AbSeq, Sample Tag libraries. Poly-T Template Switching Oligo (TSO) was added during reverse transcription reaction to allow identification of VDJ recombination events in B and T cells. This was followed by denaturation, hybridization, Klenow extension (New England Biolabs, #M0212L) and treatment with Exonuclease-I. cDNA Libraries were prepared in a two-step nested PCR reaction followed by Index PCR using BD Rhapsody Targeted mRNA and AbSeq Amplification Kit (BD Biosciences, #633774) and BD Rhapsody™ Immune Response Panel Hs (BD Biosciences, #633750). We included BD Rhapsody Supplemental Panel (BD Biosciences, #633742) to look at additional genes GATA3, MAF, CCR6, CD40L, TOX, IL6R, IL9R and IL10. After the first PCR, longer PCR products (mRNA, TCR, BCR) were separated from shorter products (AbSeq & Sample Tag) based on double-sided size selection using Agencourt AMPure XP beads (Beckman Coulter, #A63880). The concentration of libraries was estimated using Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, #Q32851). The products from second PCR were diluted as per protocol's recommendations for Index PCR. The quality check and quantification of Index libraries was performed using Agilent 2200 TapeStation with High Sensitivity D5000 ScreenTape (Agilent).