OEX022989
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OEX_Shen_2304051419 |
Transcriptomic |
The BALF was passed through a 100 μm filter (Falcon,3523260) and centrifugated (400g, 10 min, 4℃). Pelleted cells were resuspended in red blood cell lysis buffer (Solarbio, R1010) and incubated for 2 min, then passed through a 40 μm filter (Falcon,3523240), collected by centrifugation (400g, 10 min, 4℃) and resuspended in PBS (BI, 02-024-1ACS) containing 0.04% BSA (Sigma, B2064). Cells were manually counted by Trypan blue (Thermo, T10282) and AO-PI (LUNA, D23001) after each centrifugation (400g, 10 min, 4℃) and resuspended. Single cells were processed using Chromium Controller (10X Genomics) according to the manufacturer's protocol.
By using Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics, 1000268) and Chromium Next GEM Chip G Single Cell Kit (10x Genomics, 1000120), we performed single cell 3' gene expression profiling. The cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Cell-barcoded 3' gene expression libraries were sequenced on an lllumina NovaSeq6000 system by Shanghai Biochip Co., Ltd., Shanghai, China. |
2023-04-05 |