GSM8385298: CM5, PBMC; Homo sapiens; RNA-Seq
At each sample collection site, PBMCs were separated from the whole blood using density gradient centrifugation, and PBMC and BAL samples were cryopreserved in liquid nitrogen as separate samples for single-cell experiments. The PBMCs were cryopreserved in 1.5 ml cryotubes using 10% DMSO in heat-inactivated FBS and were stored long-term in liquid nitrogen. Initial PBMC sample collection, processing, and storage did not differ significantly among the three patient sample collection sites. In Mexico City, bronchoalveolar lavage samples were collected and stored long-term in liquid nitrogen using 10% DMSO in heat-inactivated FBS. All patient samples were shipped for processing in New Haven, CT, to minimize technical and batch effects arising from sample processing. Samples were randomized based on age, sex, diagnosis, and smoking history into different batches for sample preparation and single-cell library construction. Sixteen samples were processed at a time to reduce batch effects. The sample preparation protocol used was modified from 10X Genomics with the following modifications. Cryopreserved samples were thawed in a 37 °C water bath, and 1.5 ml of 10% FBS in Dulbecco's Modified Eagle Medium (DMEM) was added to each tube. The cells were centrifuged, the supernatant was removed, and the cells were resuspended in D-PBS with 0.04% bovine serum albumin (BSA). The sample was passed through a 40 µm cell strainer into a 2-mL microcentrifuge tube. The cell concentration was determined using Trypan Blue solution, and Invitrogen Countess Automated Cell Counter. Samples with isolated single cells were diluted, and these cells were loaded into the chip with an anticipated recovery of 10,000 cells according to the manufacturer's protocol for Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) (from samples from New Haven and Mexico City only). To further reduce batch effects, Chicago samples were pooled, such that each chip lane was loaded with two or three patient samples, with an anticipated recovery cell of 20,000 cells to 30,000 cells, respectively. Any leftover samples were fixed in 80% methanol and stored at -20C to preserve nucleotide integrity for downstream DNA extraction and whole-exome sequencing to SNP-demultiplex mixed patient samples from Chicago. All subsequent library construction steps were followed precisely, and all cDNA library QC was performed using an Agilent 2100 Bioanalyzer. The single-cell cDNA libraries were sequenced using an Illumina HiSeq4000 machine at the Yale Stem Cell Genomics Center (samples from New Haven and Mexico City) and from an Illumina NovaSeq X at the Yale Center for Genomic Analysis (samples from Chicago). Samples were sequenced at a depth of 150 million reads per sample to allow for adequate transcriptome capture.