title: GSM8602665: FlareTSLP+cells in the distal small intestine, replicate 2, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Intestine tissues were harvested and flushed with ice-cold PBS. After removing the Peyer's patches, gut tissues were opened and washed with PBS to remove lumen content and mucus.  Segments were incubated while rocking for 15 min at 37 °C in 20 ml HBSS (Ca2+- and Mg2+-free) containing 5% FCS, 10 mM HEPES, 10 mM EDTA and 10 mM dithiothreitol. Tissue segments were vortexed vigorously for 30 s to remove the epithelial fraction. The remaining tissue segments were incubated a second time while rocking for 15 min at 37 °C in 10 ml of the same HBSS/FBS/EDTA/DTT media and then vortexed for 30 sec. The tissue segments were transferred to a new tube and incubated while rocking for 5 min at 37 °C in 20 ml HBSS (with Ca2+ and Mg2+) containing 2% FCS and 10 mM HEPES before transferring to new tubes containing 5 ml HBSS (with Ca2+ and Mg2+), 3% FCS, 10 mM HEPES, 30 µg/ml DNase and 0.1 unit/ml Liberase TM, followed by processing with gentleMax Octo Dissociator program “intestine”. Lamina propria cells were pelleted, washed and filtered with 70 µM cell strainers before staining for flow cytometry.  Standard surface and/or intracellular staining protocols were used followed by DAPI staining for dead cell exclusion. Samples were processed using BD FACSAria™. Live cells were purified via FACS based on tdTomato expression and subsequently labeled with unique barcoded H-2/CD45 TotalSeq-B anti-mouse hashtag antibodies for multiplexing. The cells were processed according to the manufacturer's instructions using the Chromium Next GEM Single Cell  3′ Kit v3.1. Briefly, cell counts were performed using a hemacytometer, and the desired number of cells was injected into microfluidic chips to create Gel Beads-in-Emulsion (GEMs) with the 10X Chromium controller. Reverse transcription (RT) was performed on the GEMs, followed by purification and amplification of the RT products. Cell multiplexing oligo (CMO) DNA was separated from cDNA using size selection with SPRIselect beads. Gene expression (GEX) libraries and CMO libraries were generated separately and profiled using the Bioanalyzer High Sensitivity DNA kit. Prior to sequencing, the GEX and corresponding CMO libraries were mixed in a 4:1 ratio. Sequencing of the mixed libraries was conducted on the Illumina NovaSeq X platform.