GSM5708779: Intestine control (WT); Mus musculus; RNA-Seq
For bone marrow samples, mouse femurs were cleaned with alcohol, washed with ice-cold PBS containing antibiotics, and the metaphysis removed. The marrow was flushed with buffer (2 mM EDTA + 0.5% BSA in PBS) using a 23G syringe. The cells were gently mixed, filtered through a 40-mm cell strainer, and centrifuged at 300 g for 10 min. The pellets were resuspended in 0.04% BSA-PBS and counted using trypan blue. A total of 1 x 105 cells were used for the analysis (1000 cells/mL). For the intestinal samples, 2 cm of intestine above and below cecum, excluding cecum, were excised and flushed with ice-cold PBS containing gentamycin using a 20G needle. The intestine was placed in a dish with fresh PBS and cut open longitudinally. The walls were washed to remove any residual matter. The intestine was incubated in ice-cold PBS containing 10 mM EDTA (10 mL) at 4⁰C with gentle rocking for 30 min. The tissue was into ice-cold PBS and shaken with vigorous bursts to release the epithelial cells. The supernatant was centrifuged at 300 g for 5 min at 4⁰C. The pellet was washed once with ice-cold PBS, resuspended in PBS containing 1 mg/mL collagenase, and incubated at 37⁰C for 5 min. The tube was then placed immediately on ice, followed by the addition of 5% (v/v) FBS. The cell suspension was centrifuged, washed with PBS, and filtered through a 40-mm cell strainer. The cells were resuspended in 0.04 % BSA-PBS and counted using trypan blue. A total of 1 x 105 cells were used for the analysis (1000 cells/mL). Single cell libraries are generated using the 10X Genomics Chromium Single Cell 3' Reagent V3 Kit according to manufacturer's protocol.  Cell suspensions are first assessed with Trypan Blue using a Countess FL automated cell counter (ThermoFisher), to determine concentration, viability and the absence of clumps and debris that could interfere with single cell capture.  Cells are loaded into the Chromium Controller (10X Genomics) where they are partitioned into nanoliter-scale Gel Beads-in-emulsion with a single barcode per cell.  Reverse transcription is performed and the resulting cDNA is amplified.  The full-length amplified cDNA is used to generate gene expression libraries by enzymatic fragmentation, end-repair, a-tailing, adapter ligation, and PCR to add Illumina compatible sequencing adapters.  The resulting libraries are evaluated on D1000 screentape using a TapeStation 4200 (Agilent Technologies), and quantitated using Kapa Biosystems qPCR quantitation kit for Illumina. They are then pooled, denatured, and diluted to 300pM with 1% PhiX control library added.  The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge and sequenced on a NovaSeq6000 following the manufacturer's recommended protocol (Illumina Inc.).