title: GSM8714176: Thymus_scRANseq_HTO; Mus musculus; RNA-Seq
library_construction_protocol: Mice were anesthetized with intraperitoneal injection of sodium pentobarbital (100 mg/kg) and perfused transcardially with PBS, each organ was removed. For the preparation of brain cells, brain parenchyma was homogenized with a potter tissue grinder in RPMI containing FBS (2% v/v), 15 mM HEPES buffer and 0.54% glucose (Modified from Masuda et al. Nat Immunol. 202037). After straining and centrifugation, the pellet was suspended in 37% Percoll (Cytiva, 17-0891-01) diluted with RPMI and cells were isolated by centrifugation at 800 × g for 20 min at 4 °C with slow acceleration and deceleration. The pellet was collected, washed with RPMI supplemented with FBS (2% v/v), and stained for flow cytometry. For the preparation of dura cells, the dura mater was removed from the skull under a microscope and incubated with 1 mg/ml collagenase VIII (Sigma-Aldrich, C2139) and 0.4 mg/ml DNase I (Roche, 10104159001) at 37 °C for 20 minutes. Digested dura mater was disrupted by pipetting, filtrated through a 70 μm cell strainer and washed with 2% FBS/RPMI. For the preparation of intestinal cells, the feces in the colon and small intestine were washed out and cut into 1 cm pieces. Peyer's patches were cut from the small intestine under a microscope, grinded on a strainer, and cells were isolated by washing with 2% FBS/RPMI. Intestinal fragments were washed with PBS and incubated in 30 mM EDTA/PBS for 8 min at RT. After washing with PBS, they were further minced into small pieces and incubated with 1 mg/ml Collagenase D (Roche, 11088882001) and 0.1 mg/ml DNase I at 37 °C for 30 min. The tissue was digested by pipetting, and the supernatant was collected through a strainer. Then, 1 mg/ml Collagenase D was added and incubated at 37°C for 15 minutes, and the step of collecting the supernatant was repeated twice, and then all intestine was disrupted by pipetting and filtrated through a cell strainer. After centrifugation, cells were suspended in 36% Percoll diluted with RPMI, 72% Percoll diluted with PBS was injected into the bottom and centrifuged at 800 × g for 20 min at 25 ºC with slow acceleration and deceleration. A cell layer at the interface was collected and washed with RPMI supplemented with FBS (2% v/v) and stained for flow cytometry. For the preparation of lung cells, lungs were minced and incubated with 2 mg/ml CollagenaseD and 0.1 mg/ml DNase I at 37°C for 20 minutes. After the reaction, the supernatant was collected through a strainer, and the step of adding 2 mg/ml Collagenase D and 0.1 mg/ml DNase I to the precipitate again, reacting at 37°C for 20 minutes, and collecting the supernatant was repeated twice. After centrifugation, cells were suspended in 36% Percoll diluted with RPMI, 72% Percoll diluted with PBS was injected into the bottom and centrifuged at 800 × g for 20 min at 25°C with slow acceleration and deceleration. A cell layer at the interface was collected and washed with RPMI supplemented with FBS (2% v/v) and stained for flow cytometry. Spleen and thymus were filtrated through a 40 µm cell strainer. CD45+CD3ε+TCRγ/δ+TCR-Vγ6+ were sorted by FACSMelody (BD Biosciences). Libraries were prepared according to the protocol of Chromuim Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) (10x Genomics, PN-1000263) using a Chromium controller (10x Genomics). Briefly, GEMs were first generated by Master Mix with barcoded Single Cell VDJ 5' Gel Beads, cells and distribution to Oil to Chromium Next GEM Chip K (10x Genomics, PN-1000286). Barcoded cDNA was then generated from the GEM. This cDNA was amplified by PCR reaction to generate, 5' Gene Expression library and Feature Barcode library (10x Genomics, PN-1000190, -1000256, -1000215, and -1000250). Sequencing was then performed by Novaseq 6000 (Illumina).