title: GSM8193186: BALF, X771100, replicate 2, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Bronchoalveolar lavage was operated by virtue of wedging the bronchoscope into a subsegmental bronchus. Lavage was aspirated with 20 mL saline solution. One of the two equal aliquot each sample were resuspended in cooled RPMI 1640 and passed through a 70-μm nylon strainer to remove clumps, debris and phlegm three times. After the lysis of red blood cells, the aliquot was centrifuged to precipitate cells, resuspended and then counted using the Countstar viable cell counter. Dead cells were removed with low-speed centrifugation before single-cell capturing. Single-cell suspensions (2×105 cells/mL) with PBS (HyClone) were loaded onto microwell chip using the Singleron Matrix® Single Cell Processing System. Barcoding Beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the Barcoding Beads and to obtain cDNA, and PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kits (Singleron) . Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads.