GSM8385325: IPFE10, 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'