title: GSM4729129: E17.5 WT1 scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Isolated tissues were taken immediately to a sterile laminar flow tissue culture hood where they were, rinsed in fresh PBS and then minced finely using a scissors in a 100mm2 Petri dish. The minced lung tissue was then suspended in a digestion media containing 0.125% vol/vol Trypsin-EDTA (#25300056, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1mg/ml Bovine Serum Albumin (#15260037, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/ml DNase 1 (#DN25, Sigma Aldrich, St. Louis, MO, USA), 1 mg/ml Collagenase IV (#LS004209, Worthington Biochemical, Lakewood, NJ, USA)  and transferred a tissue culture incubator at 37C for 30 minutes. At 10-minute intervals the lung digestion solution was titurated 10 times using a 10 ml glass pipette. Following the incubation period, the supernatant and remaining tissue was passed through a 100 m strainer into a 50ml tube. The strainer was washed with Dulbecco's Modified Eagle Medium (#11995065, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% vol/vol fetal bovine serum (#SH3062601, HyClone, GE Healthcare, Chicago, IL USA). The tube was then centrifuged at 1600 rpm for 10 minutes at 4C and the pellet resuspended in Hank's Buffered Saline Solution (#14175095, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 0.2 mM EGTA, 10mM HEPES (#15630106, Gibco, Thermo Fisher Scientific, Waltham, MO, USA), 2% vol/vol FBS and 1% vol/vol antibiotic-antimycotic (#15240062, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) referred to hereafter as HBSS+. Red blood cells were preferentially lysed by treating the isolated cells with 1X RBC lysis buffer (# 00-4333-57, eBiosceinces, Thermo Fisher Scientific, Waltham, MA, USA) for 45 seconds, followed by immediate dilution in 20 ml HBSS+. Cells were centrifuged again and resuspended in fresh HBSS+ prior to florescence-activated cell sorting (FACS). 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.