Unknown,Transcriptomics,Genomics,Proteomics

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Single cell RNA-sequencing of T cells to examine barcode swapping

ABSTRACT: T cells from OT-I mice were stimulated with 4 different peptide ligands for 6 hours and sorted into two 96-well plates. Cells on each plate were barcoded using a mutually exclusive 8-by-12 set of indexes, such that indexes present on plate 1 were completely absent from plate 2 and vice versa. Libraries from each plate were pooled in equimolar quantities and sequenced on an Illumina HiSeq 4000. Libraries were demultiplexed allowing for all pairs of indexes, including the expected combinations (pairs of barcodes used within each plate) and unexpected combinations (pairs containing one barcode from each plate). This was repeated after sequencing the same pool of libraries on the HiSeq 2500.

INSTRUMENT(S): Illumina HiSeq 2500, Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Arianne Richard

PROVIDER: E-MTAB-6843 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To probe the phenotype of the cDC compartment during pathology, we developed a simplified human experimental skin blister model to sample infiltrating cells in order to examine the function and kinetics of DC populations in response to a bacterial insult in healthy individuals. ASDC were recruited to the inflammatory site, displaying a distinctive effector signature. Human Skin Blister 1.5 x 107 UV killed E.coli (Strain: NCTC 10418, Source: Public Health England, UK) in 100 µl sterile saline was intradermally injected into the forearm approximately 7 cm from the cubital fossa. For the suction blister, a 10 mm diameter suction blister was induced at 24 hours post challenge over the injection site by a negative pressure instrument. Once a blister formed, blister exudate was collected, cells were isolated. Next, Blister cells were stained for DC subsets and index sorted on FACS ARIA III (BD Biosciences) into 96 well plate containing lysis buffer, one cell/well - using the gating strategy in Supplementary Figure 1a. Plates were sealed and stored at -80°C. Single-cell cDNA libraries were prepared using the SMARTSeq v2 protocol (Picelli et al., 2014) with the following modifications: (i) 1 mg/mL BSA Lysis buffer (Ambionâ Thermo Fisher Scientific); and (ii) 200 pg cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, USA). All samples were subjected to an indexed paired-end sequencing run of 2x151 cycles on an Illumina HiSeq 4000 system (Illumina, San Diego, CA, USA), with 300 samples/lane. Pre-processing, quality assessment and control and analysis of SMARTseq2 single cell transcriptome data. Paired-end raw reads were aligned to the human reference genome (GRCh38 version 25 release: Gencode) using RSEM version 1.3.0. Transcript Per Million read (TPM) values were calculated using RSEM and used for downstream analysis. Quality control, selection of highly variable genes, PCA, and differential gene analysis was performed using the Seurat R package. The expression levels of key signature genes by known cell types was used to annotate the cell clusters accordingly.

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Single cell RNA-sequencing of T cells to examine barcode swapping

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