GSM8693835: TCRab+ ZsG- IELs; Mus musculus; RNA-Seq
Whole small intestine was isolated from mice and kept on media (RPMI supplemented with 3% FBS, 1% L-glutamine, 1% Pen/Strep, 50uM beta-mercaptoethanol). After flushing the intestine, Peyer's patches were removed, and tissue tragments were agited on a plate stirrer at 37C 5% CO2 in serum-free media containing EDTA and DTT. Cell suspensions were filtered and washed with media, then counted on a hemocytometer. Cells were stained for CD45, TCRb, TCRd, and DAPI prior to sorting by the BD FACSAria Fusion instrument. Single cell gene expression profiling was performed using the Chromium Next GEM Single Cell 3ʹ v3.1 (Dual Index) kit. Each cell suspension was loaded onto a well of Chip G on the 10x Genomics Chromium Controller System following the manufacturer's user manual (10x Genomics). Barcoding and cDNA synthesis were performed according to the manufacturer's instructions. Qualitative analysis of cDNA was performed using the 2100 Agilent Bioanalyzer High Sensitivity assay. The cDNA libraries were constructed using the 10x Chromium Single cell 3' Library Kit v3.1 (dual index) according to the manufacturer's protocol. Quality assessment of final libraries was done on Qubit fluorometer using a DNA High Sensitivity assay (Thermo Scientific) and a 2100 Agilent Bioanalyzer High Sensitivity assay (Agilent Technologies). Libraries were sequenced on an Illumina NextSeq 500 using the NextSeq 500/550 Mid Output Kit v2.5 (150 Cycles) sequencing kit, with the following read length: 28 bp Read1 for the 10x cell barcode and UMI, 90 bp Read 2 for the insert, and 10 bp I7 and I5 for the sample index. Phix (Illumina) was spiked in at 1% as per kit manual recommendation (10x Genomics).