GSM6248991: Batch 3 PBMC 10x Lane 10 (Batch3P10) GEX; Homo sapiens; OTHER
Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human 'hashtag' antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. Hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 137 surface proteins and SARS-CoV-2 S1 protein probe. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. PBMC samples were partitioned into single cell Gel-Bead in Emulsion (GEM) mixed together with the reverse transcription (RT) mix using 10x 5' Chromium Single Cell Immune Profiling Next GEM v2 chemistry (10x Genomics, Pleasanton, CA), as previously described (PMID: 33713619). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression and cell surface protein libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5' Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the following sequencing parameters: read1-100-cycle, i7-10-, i5-10, read2-100. single cell RNAseq (10x Genomics 5' v2)