GSM3557941: T5_Expression; Homo sapiens; RNA-Seq
The BALF samples were filtered with double-layer gauze and wash tiwce with PBS containing 0.5% FBS and 0.5mM EDTA. Then the cell pellets were mixed and stained with mutiple antibody combinations (CD45 PE dazzle594, CD14 FITC, CD16 FITC, CD3 BV711) for 30 mins before wash with PBS containing 0.5% FBS and 0.5mM EDTA. Immune cells were sorted by FACS Aria Sorp system. Single cell solution (viability >90%, 1000 cells/uL) was prepared and partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs). This would result with expected mRNA barcoding for ~5000 single-cells and multiplet rate of ~3.9%. After reverse transcription, GEMs were broken and barcoded-cDNA purified with Silane magnetic beads and Solid Phase Reversible Immobilization (SPRI) beads. Barcoded-cDNA was amplified by PCR (98ºC for 45s; [98ºC for 20s, 67ºC for 30s, 72ºC for 1 min] x 14; 72ºC for 1 min).The cDNA was splited into two subsamples subjected to library construction according to the instructions of Chromium Single Cell 5' library and TCR- or BCR-enriched library,respectively.
GEO Accession
GSM3557941