GSM8947757: WT_PBS; Mus musculus; RNA-Seq
The fresh lung tissues were cut into approximately 1mm3 pieces and washed with 4mL cold PBS solution 3 times. Then the samples were minced into small pieces, and enzymatically digested using the MACS Tumor Dissociation Kit (Miltenyi Biotec) for 30 min with agitation, according to the manufacturer's instructions. The dissociated cells were subsequently passed through a 70-μm and 40-μm cell-strainer (Miltenyi Biotec) and centrifuged at 300g for 10 min. After removing the supernatant, the pelleted cells were suspended in red blood cell lysis buffer (Tiangen Biotech#RT122-1) and incubated at 4°C for 10 min to lyse red blood cells. The cell pellets were re-suspended in washing buffer (0.04% bovine serum albumin [BSA] in Dulbecco's phosphate-buffered saline [DPBS]) after washing two times with washing buffer. The final cell concentration was adjusted to 1,000 cells/μL (viability≥80%) in all samples by using Countess™ 3 Automated Cell Counter (Life). DNBelab C Series High-throughput Single-Cell RNA Library (940-001818-00) was utilized for scRNA-seq library preparation. In brief, Briefly, the cells were diluted to a concentration of 1,000 cells/mL and loaded into the cell reservoir of the microfluidic chip. Barcoded beads and droplet-generation oil were successively added to the beads and oil reservoirs. Encapsulated droplets were generated and collected using a DNBelab C4 /DNBelabTaiM4 system. Beads that captured the mRNA were recovered for reverse transcription (RT). Following polymerase chain reaction (PCR) amplification by polymerase chain reaction, complementary DNA (cDNA) was purified and quan-tified using a Qubit dsDNA kit (Thermo Fisher Scientific). Libraries of 3'-end transcripts were subsequently constructed through cDNA fragmentation, size selection, end repair and A-tailing, adapter ligation, PCR for indexing libraries, and cyclisation of sequencing libraries, according to the manufacturer's instructions. Sequencing libraries were purified and quantified using the Qubit ssDNA kit (Thermo Fisher Scientific) and Qsep100 (Bioptic).