GSM9625272: T146_B, T123_B, C155_B, C155_F, C106_F; Homo sapiens; RNA-Seq
All sample processing, flow cytometry staining and preservation of samples for subsequent 525 analyses was performed on the day of bronchoscopy. Samples were filtered to remove mucus and debris by pipetting through 70 um 529 cell strainers, before pelleting by centrifugation at 500 x G for 7 minutes at 4oC and transferring 530 supernatants in aliquots to storage at -80oC for subsequent processing. All cell pellets were 531 briefly suspended in ammonium chloride potassium (ACK) lysing buffer (Thermo Fisher 532 Scientific) to lyse erythrocytes, before washing with RPMI 1640 supplemented with 10% foetal 533 bovine serum and 5% penicillin and streptomycin (all Thermo Fisher Scientific) and 534 centrifuging at 400 x G for 5 minutes at 4oC and suspending in supplemented RPMI. Cell pellets containing 0.5-1 x 106 live cells were fixed and permeabilized using the Chromium 576 Next GEM Single Cell Fixed RNA Sample Preparation Kit (10X Genomics), as per 577 manufacturer's instructions. Briefly, cell pellets were suspended in 0.04% Ultrapure BSA 578 (Thermo Fisher Scientific) in 1X PBS (Corning), then centrifuged at 400 x G for 5 minutes at 579  17 4oC, before suspending in 10X Genomics Fixation and Permeabilization buffer, supplemented 580 with 4% molecular biology grade formaldehyde, and fixing at 4 oC for 22 hours. Fixed cells 581 were pelleted by centrifugation at 900 x G for 5 minutes, suspended in 10X Genomics Quench 582 buffer and supplemented with 10X Genomics Enhancer solution and 50% molecular biology 583 grade glycerol (Sigma-Aldrich), to a final glycerol concentration of 10%, before storing at -584 80oC until subsequent batch processing. Sample were stored for between 7 and 574 days 585 before running scRNA-seq. Fixed samples were processed in pooled scRNA-seq runs of up 586 to 10 samples using the Chromium Fixed RNA Profiling Kits for Multiplexed Samples, splitting 587 each sample over one to three barcoded probe sets, to maximise cells recovered per sample 588 where input cell number was sufficient for splitting over multiple barcoded probe sets.