title: GSM4567342: WT_ ileal LPL and stromal cells; Mus musculus; RNA-Seq
library_construction_protocol: The distal ileum of adult WT and KO mice (separately housed) were dissected and briefly rinsed in ice-cold PBS (Fisher Scientific, SH30256LS). The tissue was opened longitudinally, rinsed in ice-cold PBS, and further sliced to 1-2 mm pieces. The pieces were rinsed in 30mL of ice-cold PBS by inverting the falcon tube 10-15 times. The pieces were then transferred into fresh 30ml of ice-cold PBS. This step was repeated until the solution remained clear after inverting. Each piece was then transferred to 30 mL crypt isolation buffer, containing 5 mM EDTA (Invitrogen, Catalog No.AM9260G), 2% BSA (Sigma, Catalog No.A3294), and HBSS Ca/Mg-free (Sigma, Catalog No.H9394), and allowed to shake at 37˚C for 15 minutes. The tubes were subsequently vigorously shaken to release the epithelial layer from the pieces after which the solution was discarded. This was repeated in another 30ml of crypt isolation buffer. The pieces were then incubated in DMEM/F12 medium (ThermoFisher, 12634-010) for 10 minutes on a petri dish, at room temperature. The solution was discarded, and the pieces were thoroughly minced. 10ml of Hank's balanced salt solution with calcium and magnesium (Sigma, Catalog No.55037C) containing 100 U/mL Collagenase II (ThermoFisher, Catalog No.17101015), 500 U/mL DNase I (Sigma,  Catalog No.DN25), was added to the minced tissue and allowed to shake at 37˚C for 30 minutes. When tissues completely digested, the solution was passed through a 70µM cell strainer into a 50mL falcon tube. The tubes were centrifuged at 200g for 10 minutes. The pellets were resuspended in 1mL of FACS solution (2% BSA in PBS). The single-cell suspensions were stained with DAPI and immediately subjected to sorting by BD Biosciences Aria II Flow Cytometer (BD FACSAria II). Single viable lamina propria lymphocytes and mesenchymal cells were gated by forward scatter, side scatter and by negative staining for DAPI.  Cell number and viability were determined by a Propidium Iodide based fluorescence assay using Moxi GO II System, Orflo Prod#MXG102 (ORFLO Technologies, LLC). Droplet-based single-cell partitioning and single-cell RNA-Seq libraries were generated using the Chromium Single-Cell 3′ Reagent v3 Kit (10X Genomics, Pleasanton, CA) on the 10X Chromium Controller as per the manufacturer's protocol. Live cells in single cell suspension with 95% viability were mixed with Gel beads, RT reagents and Partitioning Oil into a Single-Cell 3′ Chip and loaded onto 10X Chromium Controller for GEM generation. Briefly, the protocol includes RT, cleanup, cDNA amplification, fragmentation, end repair & A-tail prep, adapter ligation and incorporation of sample indices into finished libraries, which are compatible with Illumina next-generation sequencing platforms. Sample quantification and quality control were determined using Qubit Fluorometer (Invitrogen, Life Technologies) and TapeStation (Agilent Technologies, Santa Clara CA) respectively. cDNA libraries were sequenced on Illumina NovaSeq 6000 sequencer (Illumina, San Diego, CA) with a configuration of 28/8/0/91-bp for cell barcode, sample barcode and mRNA reads respectively as recommendation by 10X Genomics.
attributes.tag: GEO Accession
attributes.value: GSM4567342