title: GSM5212975: Wildtype_atrophy_rep1; Mus musculus; RNA-Seq
library_construction_protocol: Intestinal tissues were incubated in 30mM EDTA, first on ice for 20min with 1.5mM DTT and second at 37C for 10min without DTT, and then shaken to lift the epithelium. Epithelial cells were dissociated in an enzymatic cocktail containing 1mg/mL Collagenase/Dispase (Roche) and 0.2mg/mL DNase I (Roche) at 37C for 10min with intermittent shaking. The cell suspension was additionally subjected to vigorous pipetting and filtered through 70 and 40um strainers. Cells were washed in 10% FBS/PBS solution and resuspended in FACS buffer containing 2% BSA, 2mM EDTA, and 25mM HEPES in PBS. ROCK inhibitor (R&D Systems) was added to every solution throughout the experiment. After excluding debris and gating for singlets, live (SYTOX-Red negative) epithelial cells were sorted and resuspended in 10% FBS/DMEM-F12 solution for single-cell capture and sequencing. Single cell suspensions went through GEM generation and barcoding, followed by GEM RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads. Samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3' Reagent Kits (v3 Chemistry) user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium Single Cell 3' GEM, Library and Gel Bead Kit v3 (PN-1000075), Chromium Single Cell B Chip Kit (PN-1000153), Dual Index Kit TT Set A (PN-1000215) and Chromium i7 Multiplex Kit, 96 rxns (PN-120262) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 28x10x10x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library.
attributes.tag: GEO Accession
attributes.value: GSM5212975