GSM8693206: Citeseq, MLN, GEX + ADT; Homo sapiens; OTHER
Cell suspensions from fresh MLNs draining the inflamed colon were prepared by shredding MLNs as previously described26 and immediately cryopreserved in FCS, DMSO 2%.Frozen MLN samples from UC (n=3) and CD (n=3) were thawed. Cell count for viability was performed by Trypan blue exclusion and 3.5 x 106 cells from each sample were labelled with Sample Tags using the BD Single-Cell Multiplexing Kit according to the manufacturer's protocol. After tagging, cells were pooled and labelled with FACS antibodies (Table S2) before sorting. FACS-sorted CD3+ T cells and CD19-CD3- non-B, non-T cells were pooled at a 1:2 ratio. Next, the bar-coded pooled sample was labeled with nine BD Abseq antibodies (2μl/antibody; Table S3) according to the BD protocol, washed twice and resuspended in cold BD Sample Buffer for a total of 45,000 cells in 650 μL. For whole transcriptomics experiment, the BD Rhapsody Express system was used.Cell count was performed on the BD Rhapsody Express system to check for viability before cell loading for single cell capture and cDNA synthesis. After priming the nanowell cartridge, the pooled sample was loaded onto one BD Rhapsody cartridge and incubated at room temperature for 20 minutes. Preparation, loading and washing of Cell Capture Beads, cell lysis and retrieval of Cell Capture Beads was performed according to the BD protocol. Quality control (QC) analysis by the BD Rhapsody scanner indicated a total of 8489 wells with viable cells and a bead, and a cell multiplet rate of 2%. Libraries were prepared for 3' whole transcriptome (WT) analysis, AbSeq and Sample Tag according to the BD mRNA WTA, AbSeq and Sample Tag library preparation protocol. Index PCRs for WTA mRNA, AbSeq and Sample Tag were performed to prepare final libraries for sequencing. After library preparation and indexing steps, PCR product cleanup was done using the Agencourt AMPure XPbeads (Beckman Coulter Life Sciences) with bead and PCR product ratios recommended by BD. QC control before sequencing was performed on the 2100 Agilent Bioanalyzer. Sample bioanalyzer trace showed PCR product size and peak in the expected range for the three libraries. Sequencing was performed at The Centre for Applied Genetics (Hospital for SickKids, Toronto, Canada). The libraries were pooled for paired-end (100bp) sequencing on the NovaSeq Illumina platform with 40,000 reads/cell for WT mRNA, 120 reads/cell for Sample Tag and 9000 (9 AbSeq x1000) reads/cell for AbSeq libraries.