Illumina HiSeq 4000 paired end sequencing; Single Cell RNAseq from IL6R deficient patient and healthy parent
Single Cell RNAseq from IL6R deficient patient and healthy parent
Fresh PBMCs isolated over a Ficol gradient. Cell suspensions of both samples were resuspended in PBS-0.04% BSA and loaded into Chromium microfluidic chips with 10x Genomics 5' V(D)J and gene expression chemistry to generate single-cell gelbead emulsions using the Chromium controller (10X Genomics) according to the manufacturer recommendations.Single cell RNA-seq libraries were prepared in CRUK CI Genomics Core Facility using the following: Chromium Single Cell 5′ Library & Gel Bead Kit PN-1000006, Chromium Single Cell A Chip Kit PN-1000009, Chromium i7 Multiplex Kit PN-120262, Chromium Single Cell V(D)J Enrichment Kit, Human B Cell PN-1000016, Chromium Single Cell V(D)J Enrichment Kit, Human T Cell PN-1000005 (10x Genomics, Pleasanton, CA) and the Single-Cell V(D)J Reagent Kits User Guide (Manual Part CG000086 Rev J). Suspensions containing ∼16k cells were loaded on the instrument with the expectation of collecting up to 9,000 gelbead emulsions containing single cells (expected multiplet rate ~7%). RNA from the barcoded cells for each sample was subsequently reverse-transcribed in a T100 Thermal cycler (Bio-Rad) and all subsequent steps to generate single-cell libraries were performed according to the manufacturer's protocol with no modifications with 14 cycles used for cDNA amplification. Then ~50 ng of cDNA were used for gene expression library amplification by 14 cycles in parallel with cDNA enrichment and library construction for T and B cells libraries.  Library quality was confirmed with Agilent TapeStation 4200 (High Sensitivity D1000 ScreenTape to evaluate library sizes) and BMG LABTECH Clariostar Monochromator Microplate Reader (Invitrogen Quant-iT dsDNA Assay Kit, high sensitivity to evaluate dsDNA quantity). Each sample was normalised to equal molar concentration, pooled in the following ratio: 90% gene expression library, 5% Human B cell library, 5% Human T cell library and sequenced on an Illumina HiSeq 4000 as 2 × 150 paired-end reads, one full lane per pool (gene expression data were trimmed to 26bp read 1, 8bp i7 index, 98bp read 2 prior to analysis).