title: GSM4763854: IPF1_Expr_norm; Homo sapiens; RNA-Seq
library_construction_protocol: Freshly isolated human lung tissues were obtained from Cedars-Sinai Medical Center and UCLA and were dissociated using a standard protocol in our laboratory64. Lung tissue was taken to a sterile tissue culture hood, transferred to a Petri dish and rinsed in PBS. Airways >2 mm were resected from the surrounding tissues and discarded along with the visceral pleura. The remaining tissue was finely minced with a scissors and then a straight razor blade. The minced lung tissue was then washed in Ham's/F12 media (#11320033, Gibco, Thermo Fisher Scientific, Waltham, MO, USA) at 4C for 20 minutes to remove blood and then centrifuged at 600 rpm for 5 minutes in a pre-cooled centrifuge. The media was removed, and the tissue transferred to a 50 ml conical tube containing 2 mg/ml Dispase II (Thermo Fisher Scientific, Waltham, MA, USA) in DMEM/F12 overnight at 4C with gentle agitation. The next day the suspension was heated to 37C for 30 minutes, and the centrifuged for 5 minutes at 4C. The supernatant was removed, and any large pieces of tissue finely minced again with a straight razor blade. The tissue was then titurated in a digestion media containing 10 U/ml elastase (#LS002280, Worthington Biochemical, Lakewood, NJ, USA) and incubated for 30 minutes at 37C. An equal volume of HBSS+ was then added, the solution titurated and then centrifuged at (600 g, 5 minutes, 4C). The supernatant was removed, and the tissue incubated at 37C for 15 minutes with DNase I solution. The suspension was titurated and transferred to a 70 m cell strainer over a new 50 ml tube. The strainer was rinsed three times with 10 ml HBSS+. The suspension was centrifuged (600 g, 5 minutes, 4C) and the cells resuspended in 1 X RBS lysis buffer for 2 minutes on ice, the solution diluted with HBSS then centrifuged (600 g, 5 minutes, 4C). The supernatant was removed, and the cells resuspended in appropriate solution for further analysis. mRNA from single cells sorted from lung into lysis plates was reverse transcribed to complementary DNA (cDNA) and amplified as previously described. Library preparation and sequencing were performed as described previously. Sequencing libraries for cDNA from single cells were prepared as per the Single Cell 3′ v2 Reagent Kits User Guide (10x Genomics, Pleasanton, CA, USA). Cellular suspensions were loaded on a Chromium Controller instrument (10x Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-reverse transcription (RT) was performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). GEMs were collected and the cDNA was amplified and purified with SPRIselect Reagent Kit (Beckman Coulter, Brea, CA, USA). Indexed sequencing libraries were constructed using Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems, Roche Holding AG, Basel, Switzerland). Sequencing libraries were loaded on a NextSeq500 (Illumina, San Diego, CA, USA) with a custom sequencing setting (26bp for Read 1 and 98bp for Read 2) to obtain a sequencing depth of ~200K reads per cell.
attributes.tag: GEO Accession
attributes.value: GSM4763854