GSM6067321: H1N1 Day 7, RNA-seq; Mus musculus; RNA-Seq
After perfusing PBS 20 ml through the left and right ventricular chambers, mouse lungs were minced and digested. The isolated cells were treated with red blood cell lysis buffer (Biolegend, Cat#: 420301) for 5 minutes to lyse red blood cells. After washing with PBS twice, cells were incubated with DAPI (1 mg/ml) in suspension buffer (Ca2+ and Mg2+ free PBS, 5% BSA, 2mM EDTA) at 4 ºC for 30 mins with gentle tilting. After centrifuging, the lung cells from three mice at Day 7 post-inocuation were mixed and incubated in buffer (Ca2+ and Mg2+ free PBS,10% BSA, 2mM EDTA).  The sample was subjected to single-cell fluorescence-activated cell sorting (FACS) of DAPI- cells (live ECs) in the MoFlow Astrios machine. The DAPI- lung cells were loaded into a 10x Genomics microfluidics chip and encapsulated with barcoded oligo-dT-containing gel beads using the 10x Genomics Chromium controller using v3 kit according to the manufacturer's instructions. Library of lung cells was sequenced on NovaSeq 6000 S4 (~100k reads per cell and per lane 2x150bp Paired-Read) at a depth of ~200,000 reads per cell.