GSM9433615: BLM_Tns3; Mus musculus; RNA-Seq
Lung tissues were thoroughly cleaned and dissociated into single cells using a dissociation solvent composed of 0.35% collagenase IV, 2 mg/mL papain, and 120 units/mL DNase I. The dissociation process occurred in a water bath shaker at 37 °C for 20 min at 100 rpm. Following dissociation, the reaction was halted by adding 1× PBS containing 10% fetal bovine serum (FBS), and the cell suspension was obtained by repetitive pipette gun blowing (5–10 times). The resulting cell suspension was filtered through a 70 –30 μm cell sieve and centrifuged at 300× g at 4 °C for 5 min to collect the cell precipitate. The cells were then suspended in 100 µL of 1× PBS (0.04% BSA) solution, and 1 mL of red blood cell lysate (Macs 130-094-183, 10×, USA) was added for 2–10 min at room temperature or on wet ice to remove red blood cells. After the lysis process, the suspension was centrifuged at 300×g for 5 min at room temperature, and the resulting cell precipitate was collected. Next, 100 µL of reagent for removing dead cells was added, and after the reaction, the reagent was removed. The suspension was then resuspended in 1× PBS and centrifuged at 300× g at 4 °C for 3 min. The cell pellet was resuspended in 50 mL of 1× PBS. Cell number was counted  using the blood cell counting plate or the Countstar Rigel S2(Aber Instruments Ltd). Finally, the cell concentration was adjusted to 700–1,200 cells/µL For scRNA-seq: the cell suspension was loaded into Chromium microfluidic chips with 3' (v2 or v3, depends on project) chemistry and barcoded with a 10× Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 3' v2(v2 or v3, depends on project) reagent kit (10X Genomics) according to the manufacturer's instructions. Sequencing was performed with Illumina novaseq X Plus. For the preparation of the TCR sequencing library, approximately 2μg of total RNA from each sample was utilized. This was done using the KC-DigitalTM Stranded TCR-seq Library Prep Kit for Illumina® 150, supplied by Seqhealth Technology Co., Ltd., located in Wuhan, China, and identified by the catalog number DT0813-02. The procedure was carried out in accordance with the manufacturer's instructions. The kit is designed to minimize the duplication bias during PCR and sequencing by incorporating unique molecular identifiers (UMIs), which are 8-base random sequences, to tag the pre-amplified cDNA molecules. The library products, sized between 250-500 base pairs, were selected, quantified, and ultimately sequenced using the NovaSeq platform from Illumina®.