GSM3693032: Lung_Injured GFP; Mus musculus; RNA-Seq
The lung cells were dissociated in the digestion Buffer (10mM HEPES, 150mM NaCl, 5mM KCl, 1mM MgCl2, 1.8mM CaCl2,   2mg/ml Collagenase A, 1mg/ml Dispase II, 0.5mg/ml Elastase,  1mg/ml Dnase in PBS)  using gentleMACS Dissociator program mouse tumor implant program 01.01 and then incubated  in 37 ◦C incubator for 15 mins. After incubation, the digestion process were terminated by adding an equal volume of HBSS on ice. After straining the tissue digest through a 100-μm cell strainer, the cells were centrifuged at 300g for 10 minutes 4◦C. The cell pellets were re-suspended in 5-10 mL of RT 1X RBC lysis buffer (eBioscience 00-4333-57) for 5 minutes at RT and then diluted with an equal volume of HBSS and centrifuged again to pellets. Pellets were re-suspended in appropriate volume of FACS staining buffer (2%FBS in PBS). Each sample was added primary antibodies including Ter119-450 (Thermofisher, 48-5921-82), CD45-450 (Thermofisher, 48-0451-82) and LIVE/DEAD 450 (Thermofisher, L34963) for cell sorting and incubated for 20-30 min on ice. After washing 2 times, cells were counted and then sorted by fluorescence (antibody stainings and/or EGFP) on a BD FACS Aria II cell sorter (BD Biosciences), using FACS Diva software, into Eppendorf tube containing 20 µl of 2%FBS in PBS and stored on ice until proceeding to the GemCode Single Cell Platform. Single cells were processed through the GemCode Single Cell Platform using the GemCode Gel Bead, Chip and Library Kits (V1) or single-cell suspensions were loaded onto 3′ library chips for the Chromium Single Cell 3′ Library (V2, PN-120267) according to the manufacturer's recommendations (10X Genomics). In brief, single cells were partitioned into Gel Beads in Emulsion in the GemCode instrument with cell lysis and barcoded reverse transcription of RNA, followed by amplification, shearing and 5′ adaptor and sample index attachment. An input of 1200 single cells was added to each channel with a recovery rate of roughly 300 cells. Libraries were sequenced on an Illumina Nextseq 500 at >190,000 reads per cell. Libraries were sequenced on an Illumina Nextseq 500 at >190,000 reads per cell.
GEO Accession
GSM3693032