title: GSM4929086: Batch 1 PBMC 10x Lane 6 RNA; Homo sapiens; RNA-Seq
library_construction_protocol: 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. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. ﻿Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5' Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) 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 sequencing parameters recommended by the 10x Genomics 5' v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.
attributes.tag: GEO Accession
attributes.value: GSM4929086