GSM2790885: d21_SMA+Tm+; Mus musculus; RNA-Seq
Fluorescence- activated cell sorting (FACS) experiments were performed using fresh lung preparations. Mouse lung homogenates for single-cell flow cytometry were prepared as previously described (PMID 27400124). Briefly, fresh mouse lungs were perfused with 10 ml PBS, elastase (4 U/ml; Worthington Biochemical Corporation) were injected through the trachea to inflate the lung and dissociate epithelial cells. After that, samples were cut into approximately 1-mm3 pieces and digested with DNase I (100 U/ml; Sigma). Single cell homogenates were collected after passing through cell strainers and centrifugation. Flow cytometry was used to sort Epcam-, CD31-, and CD45- MCs. Primary antibodies to CD31, and CD45, and secondary antibody anti-streptavidin were all from eBioscience (San Diego, CA). Mouse anti-EpCAM (G8.8, catalog 118215) were from BioLegend (San Diego, CA). 7-AAD was from BD Biosciences (San Diego, CA). Singlet discrimination was sequentially performed using plots for forward scatter (FSC-A versus FSC-H) and side scatter (SSC-W versus SSC-H). Dead cells were excluded by scatter characteristics and viability stains. All FACS experiments were performed on an Aria III sorter (BD Immunocytometry Systems, San Jose, CA) at the Cedars-Sinai Medical Center Shared FACS Facility and FACS data were analyzed using FlowJo software (TreeStar, Ashland, OR). Single-cell RNA-seq libraries were prepared per the Single Cell 3′ v2 Reagent Kits User Guide (10x Genomics, Pleasanton, California). Cellular suspensions were loaded on a Chromium Controller instrument (10X Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-RT were performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA). After RT, GEMs were harvested and the cDNAs were amplified, cleaned up with SPRIselect Reagent Kit.  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 (KAPA Biosystems, Wilmington, MA). Sequencing libraries were loaded on a NextSeq500 (Illumina, San Diego, CA) with a custom sequencing setting (26bp for Read 1 and 98bp for Read 2), to obtain a sequencing depth of 100K~200K reads per cell. 10X single cell RNA-seq