title: GSM4043498: 3 hour Mesenchymal single cells from APCmin mice after treatment; Mus musculus; RNA-Seq
library_construction_protocol: Open intestine longitudinally. Wash intestine 5 times with ice-cold HBSS w/o Ca2+Mg2+. Add 37.6 ml HBSS w/o Ca2+Mg2+ to 50 ml Falcon tube. Add 2.4 ml of 0.5M EDTA (10 mM). Add in the small pieces of intestine (2-3 cm pieces) with the final volume 40 ml, invert tube to mix; incubate on ice for 20 min, wash 5 times with 40 ml cold HBSS w/o Ca2+Mg2+ and incubate on ice for 20 min. Shake vigorously for 4 min. Keep the supernatant in a new tube, and add 40ml cold HBSS w/o Ca2+Mg2+. Shake vigorously for 4 min. Repeat until getting 3 tubes of supernatant. (preparation of EP) Use 40 µm cell strainer to filter supernatant. Centrifuge (Jouan CR422) the tubes at approximately 300g for 5 min at 4°C.  Remove the supernatant and add 2 ml of TryPLE Express (containing 0.05 mM NAC and 10 µM Y-27632) per sample. Incubate for 4 min in a 37°C water bath with gently shaking in hands or stirring with a P100 pipette occasionally. Place on ice for 1 min, gently shaking. Transfer dissociated samples into 40 ml ice cold ADMEM/F12 washing medium (containing 1% L-GlytaMax, 1% HEPES, 1% Penicillin/Streptomycin, 0.05 mM NAC and 10 µM Y-27632). Shake the 50-ml tube for about 30 sec to enhance the dissociation and use 70 µm cell strainer to filter supernatant. Spin down at 500g for 5 min at 4°C. Re-suspend pellet in 3ml of ice cold ADMEM/F12 washing medium, use 20 µm syringe filter to separate any remaining mass and then get the epithelial single cell. (preparation of MC) Add 10 ml ice-cold HBSS with Ca2+Mg2+. Pre-heat Liberase enzyme solution 370C for 10min. (Prepare Enzyme solution: 10 ml HBSS with Ca2+Mg2+ , 300 ul Liberase in 50 ml Falcon tube) Transfer mesenchymal intestinal preparation to pre-heated Liberase enzyme solution. Incubate 370C for 20' with occasional gentle shaking (2X/min) Cool 50 ml tube on ice for 3 min. Add 200ul DNase I (2,000 U/ml). Shake vigorously (2x/second) to dissociate mesenchymal tissue. Transfer samples to MACS commercial tubes. Use Intestine_01 program for about a minute in MACS dissociator. Transfer dissociated tissue to 50 ml Falcon tube; add 1 ml 0.5M EDTA; mix gently by shaking. Add 30 ml HBSS with Ca2+Mg2+ + 3% FBS (900 ul) immediately; mix gently by inverting. Resuspend a few times using a 30ml syringe + 16G needle. Filter tissue through 40µm cell strainer (DB) directly into a new 50 ml tube. Pre-heat 4ml of lysis buffer in a warm bath. Centrifuge at 1600 rpm (~ 600g) 40C 7 min. to pellet cells. Add 2 ml of Hemolytic lysis buffer and keep the tube in a water bath for less than 2 min. Add up to 45ml of MACS Rinsing solution + Y27632 inhibitor to stop lysis. Filter with 70um cell strainer (DB) after lysis. Centrifuge at 500xg for 5 min at 4 degree, and resuspend with HBSS without Ca2+Mg2+ + 0.4%  BSA. Dissociated cells, having been sorted in HBSS without Ca2+Mg2+ + 0.4%  BSA, were assessed for concentration and viability via a Nexelom Cellometer Auto T4.  Cells, having been deemed to be at least 60% viable, were loaded on a Chromium Single Cell Controller (10x Genomics, Pleasanton, CA), based on live cell concentration. Libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead Kit v2 (10x Genomics) according to manufacturer's directions.  Resulting short fragment libraries were checked for quality and quantity using an Agilent 2100 Bioanalyzer and Invitrogen Qubit Fluorometer.  Libraries were pooled in groups, at equal molar concentrations.  With cells captured estimated at ~420-19,000 cells per sample, libraries were sequenced to a depth necessary to achieve 8,500-175,000 mean reads per cell - ~40-340M reads each - on an Illumina HiSeq 2500 instrument using Rapid SBS v2 chemistry with the following paired read lengths: 26 bp Read1, 8 bp I7 Index and 98 bp Read2. 10X scRNA-seq
attributes.tag: GEO Accession
attributes.value: GSM4043498