GSM9630080: GEM-KO; Mus musculus; RNA-Seq
Lungs were cut into pieces and digested for 30 min with 1mg/ml type I collagenase and 0.1mg/ml DNase Ⅰ in DMEM at 37 °C. After enzymatic incubations, lungs were further dissociated over a 70-μ m nylon cell strainer (Falcon). For isolation of lymphocytes, single-cell suspensions were then separated using a 44/67% Percoll density gradient. Red blood cells were lysed with ACK buffer (140 mM NH4Cl and 17 mM Tris-base, pH 7.4).The cells were washed in PBS and resuspended in PBS supplemented with 2% w/v FBS. Reverse transcription was carried out at 37 °C for 30 min. The barcoded cDNA on CBBs was hybridized with a random primer containing read-2 SeqPrimer sequences at the 5′ end, enabling extension to form the second strand with a 3′ cell barcode. The resulting second-strand DNA was denatured off the CBBs, purified, and amplified by PCR. The cDNA product was cleaned, ligated to adapters and sample indices, and the indexed sequencing libraries were purified with SPRI beads, quantified, and sequenced on an Illumina NovaSeq.