Bronchial brushings from 12 subjects (6 never smokers and 6 current smokers) were treated with 0.25% Trypsin/EDTA for 20 minutes and stained for sorting (FACS) into 96-well PCR plates containing lysis buffer (0.2% Triton-X 100, 2.5% RNaseOUT) compatible with downstream RNA library preparation. In each 96-well PCR plate for each subject, we sorted 84 ALCAM+ cells, 11 CD45+ cells, and maintained one empty well as a negative control. Single-cell RNA-Seq of human bronchial airway cells was performed using a modified version of the CEL-Seq RNA library preparation protocol. For each of the 12 subjects, one frozen 96-well PCR plate containing sorted cells was thawed on ice and RNA was directly reverse transcribed from whole cell lysate using primers composed of an anchored poly(dT), the 5' Illumina adaptor sequence, a 6-nucleotide well-specific barcode, a 5-nucleotide unique molecular identifier (UMI), and a T7 RNA polymerase promoter. Samples were additionally supplemented with ERCC RNA Spike-In mix (1:1,000,000 dilution) for quality control. cDNA generated from each of the 96 cells per plate was pooled, subjected to second strand synthesis and amplified by in vitro transcription. Amplified RNA was chemically fragmented and ligated to the Illumina RNA 3' adapter. Samples were again reverse transcribed using a 3' adaptor-specific primer and amplified using indexed Illumina RNA PCR primers. Samples were sequenced on an Illumina HiSeq 2500 in rapid mode, generating paired-end reads (15 nucleotides for Read 1, 7 nucleotides for index, 52 nucleotides for Read 2).
GEO Accession
GSM3773119