title: GSM7790660: Female1-CytoID-Negative; Mus musculus; RNA-Seq
library_construction_protocol: Esophageal epithelium-enriched tissue layer was peeled from muscle then incubated in 1 ml 1X Dispase I (354235; Corning; Bedford, MA, USA) diluted 1:4 in Hank's Balanced Salt Solution (HBSS; 14025-076; Gibco; Grand Island, NY, USA) for 10 minutes at 37 °C with shaking at 1000 RPM (5384; Eppendorf F1.5 Thermomixer; Hamburg, Germany). Following removal from Dispase I, esophageal epithelium was minced with sharp scissors then incubated in 1 ml of 0.25% Trypsin-EDTA (25-053-CI; Corning; Manassas, VA, USA) for 10 min at 37 °C with shaking at 1000 RPM. Trypsin and tissue pieces were forced through 70 μm cell strainer into 50 ml conical tube containing 4 ml soybean trypsin inhibitor (STI; 9035-81-8; Grand Island, NY, USA). Cells were pelleted at 1300 RPM for 3 min then resuspended in 500 μl complete mouse keratinocyte–serum-free medium (37010022; Gibco; Carlsbad, CA, USA). Cell number and viability were measured using Automated Cell Count (AMQAX1000, Invitrogen Countess II, Bothell, WA, USA) by mixing 5 μl cell suspension with 5 μl 0.4% trypan blue solution (T10282; Invitrogen; Eugene, OR, USA). To isolate Cyto-ID Low and Cyto-ID High esophageal basal cells, the resulting single cell suspension was incubated at 37C for 30 minutes with Integrin-b1 antibody (APC anti-mouse/rat CD29; 102216; Biolegend) at 1:100 dilution. Cells were washed, pelleted at 1000 RPM for 5 minutes, and incubated at 37C for 30 minutes with Cyto-ID (ENZ-51031-K200; Enzo Life Sciences) at 1:1000 dilution. After washing, cells were resuspended in FACS buffer (1% of BSA in PBS) and 2 μg/ml DAPI (D1306; Invitrogen) was added immediately before sorting using FACS Aria Cell Sorter (BD Biosciences). Total live esophageal epithelium cell compartment (DAPI negative; Integrin-b1 positive) was sorted based upon Cyto-ID fluorescence into two populations: Cyto-ID Low (negative)  and Cyto-ID High (positive). RNA was isolated from sorted Cyto-ID Low and Cyto-ID High murine esophageal basal cells using RNeasy Mini Kit (74104; Qiagen) according to the manufacturer's protocol. To make stranded mRNA-Seq library: 500 ng total RNA from each sample were used to make library according to the product guide of stranded mRNA library kit (, E4720L; NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina). In short, mRNAs were enriched twice via poly-T based RNA purification beads and subjected to fragmentation at 94C for 15 minutes via divalent cation method. The 1st strand cDNA was synthesized by reverse transcriptase and random primers at 42C for 15 minutes, followed by 2nd strand synthesis at 16C for 1 hour.  A single 'A' nucleotide was added to the 3' ends of the blunt fragments at 37C for 30 minutes.  Adapters with IlluminaP5, P7 sequences as well as indices were ligated to the cDNA fragment at 30C for 10 minutes.  After purification using SPRIselect Beads (B23318;Beckman Coulter), a 13-cycle PCR reaction was used to enrich fragments. PCR was set at 98C for 10 seconds, 60C for 30 seconds, and extended at 72C for 30 seconds.  Libraries were again purified using SPRIselect Beads. Quality check was performed on Agilent 2100 bioanalyzer using Agilent high sensitive DNA kit (5067-4626). Quantification was performed with Qubit 3.0 fluorometer (Q33216, ThermoFisher Scientific) using Qubit 1X dsDNA HS Assay Kit (Q33230). Sample libraries were subsequently pooled and loaded to Nextseq 500.   Samples were sequenced 50-200 bases pair-end on Nextseq 500 machine with 30 million read depth.