title: GSM9013292: scRNAseq HTO 10x Lane 4; Mus musculus; RNA-Seq
library_construction_protocol: Serum-free media was prepared with RPMI containing 20 mM HEPES,  L-Glutamine, 100U/mL Penicillin, 100ug/mL Streptomycin sulfate, 50 µM β-mercaptoethanol, 1mM sodium pyruvate, 1mM non-essential amino acids.3% media was prepared with RPMI containing 3% FBS, 20 mM HEPES,  L-Glutamine, 100U/mL Penicillin, 100ug/mL Streptomycin sulfate, 50 µM β-mercaptoethanol, 1mM sodium pyruvate, 1mM non-essential amino acids. Mice were sacrificed by CO2 inhalation. For cell extraction from small intestinal lamina propria, Peyer's patches and mesenteric adipose tissue were removed. For each intestine, tissues were cut open longitudinally, washed several times in cold PBS to remove feces, and further cut into 1- to 2-cm segments before being placed in 50 ml beakers with a stir bar. Tissue pieces were incubated in 10 ml per intestine of 3% RPMI with 5 mM EDTA, and 0.145 mg/ml DTT for 20 min at 37° C with stirring ~400 rpm. To separate the lamina propria (LP) and intestinal epithelial lymphocyte (IEL) layer, each beaker's contents were strained through a sterile fine-meshed stainless-steel sieve into a corresponding 500 ml beaker containing 10 ml of 3% RPMI media held on ice. After straining, tissue pieces from each intestine were transferred to a 50 ml conical tube containing 10 ml of serum-free RPMI with 2 mM EDTA. Each tube was shaken vigorously for 30 seconds and then strained through the respective sieve into the corresponding beaker. The shake and strain processes were repeated two more times. To isolate the IEL compartment, strained contents were filtered through 70 µm cell strainers atop 50 ml conical tubes on ice. Cell suspensions were centrifuged for 5 minutes at 450 g, 4° C. After gently discarding the supernatant, cell pellets were resuspended in 4 ml of a 30% Percoll gradient and centrifuged for 5 minutes at 1800 rpm, 4° C. After removing the supernatant with a pipette, cells were resuspended in 1 ml of 3% RPMI media. To isolate the LP compartment, small intestine pieces recovered from the shake and strain steps were placed into a 50 ml beaker and finely minced with scissors. Tissues were then incubated with 10 ml of serum-free media with 0.1 mg/ml Liberase TL and 5 mg/ml DNAse I for 25 min at 37° C with stirring ~400 rpm. After the incubation, 5 ml of 3% RPMI was added to each beaker. The contents of each beaker were pipetted through 70 µm cell strainers atop 50 ml conical tubes on ice. Each beaker and strainer was rinsed with an additional 10 ml of 3% RPMI. Cell suspensions were centrifuged for 5 minutes at 450 g, 4° C. After gently discarding the supernatant, cell pellets were resuspended in 4 ml of a 30% Percoll gradient and centrifuged for 5 minutes at 1800 rpm, 4° C. After removing the supernatant with a pipette, cells were resuspended in 1 ml of 3% RPMI media. SILP CD4^+^T-bet^+^ T cells (Live, CD45+CD90.2+, TCRb+, CD4+, T-bet-ZsGreen+) from GF T-bet-ZsGreen mice raised on casein, amino acid, or regular chow diet were sorted from samples labeled with TotalSeqC hashtags antibodies (Biolegend) using a Sony MA900 sorter. Sorted cells were pooled during sorting and 40000 cells per lane (total of 4 lanes) were loaded on a Chromium Single Cell Controller (10X Genomics) to encapsulate the cells into droplets. The gene expression (GEX), hashtag oligonucleotide (HTO) and VDJ (TCR) sequencing libraries were prepared using Chromium Single Cell 5' v2 reagent kits following the manufacturer's instructions, and sequenced on an Illumina Nextseq1000 platform (NextSeq 1000/2000 P2 reagents)