GSM8546495: IEN2; Mus musculus; RNA-Seq
Excised colons were opened longitudinally, briefly washed, and were placed in a tube containing 10mL Hanks' Balanced Salt Solution without calcium, magnesium, and phenol red (HBSS-) supplemented with 1 mM N-Acetyl-L-cysteine (NAC). The tissue was vortexed for 15 seconds, followed by a 15-second rest on ice. This was repeated for a total of 2 minutes. To collect the epithelial fraction, the rinsed colon was incubated in 10mL HBSS-, 1mM NAC, and 10mM EDTA for 2 rounds of 15 minutes each at 37C with shaking motion, followed by brief vortexing and passing through a 100um strainer. Filtered epithelial fraction was pelleted while the remaining lamina propria tissue was minced. Both fractions were further digested into single cells in HBSS- supplemented with1.5mg/mL of collagenase type VIII, 5% FBS and 25ug/mL DNase I for 25 minutes at 37C with shaking motions. The single cell suspension was achieved by filtering through a 40um strainer. Sixteen thousand cells, with a viability of over 70%, were loaded into the Chromium Controller (10X Genomics, PN-120223) on a Chromium Next GEM Chip G (10X Genomics, PN-1000120), and processed to generate single cell gel beads in the emulsion (GEM) according to the manufacturer's protocol. The cDNA and library were generated using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics, PN-1000286) and Dual Index Kit TT Set A (10X Genomics, PN-1000215) according to the manufacturer's manual. Quality control for constructed library was performed by Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and Qubit DNA HS assay kit for qualitative and quantitative analysis, respectively. The multiplexed libraries were pooled and sequenced on Illumina HiSeq 4000 sequencer with 2 × 50 paired-end kits using the following read length: 28 bp Read1 for cell barcode and UMI, and 90 bp Read2 for transcript.