GSM3391587: ScRNA-seq of airway macrophages; Mus musculus; RNA-Seq
Bronchoalveolar lavage (BAL) was performed with 10 serial lavages of 1ml PBS containing 5 mM EDTA. One animal was used for each time point. BAL cells were washed twice in PBS. FcgR was blocked using anti-CD16/CD32 for 20 minutes. For day 3 sample, neutrophils were depleted over a column by binding Ly6G prior to surface staining. Cells were incubated with primary antibody on ice for 30 minutes, washed twice, and then taken to FACS. Dead cells were excluded on the basis of scatter and DAPI exclusion. Monocytes were excluded based on size and light scattering characteristics. Antibodies and cell stains used are as follows: Anti-Ly6G (Biolegend, clone 1A8), anti-CD3 (ThermoFischer Scientific, clone 17A2), anti-CD19 (ThermoFischer Scientific, clone eBio1D3), anti-NK1.1 (ThermoFischer Scientific, clone PK136), DAPI (Biolegend, #422801), anti-CD64 (BD Biosciences, clone X54-5/7.1), anti-CD11c (ThermoFischer Scientific, clone N418), anti-CD11b (ThermoFischer Scientific, clone M1/70), and anti-F4/80 (ThermoFischer Scientific, clone BM8). FACS was performed using a BD FACSAria Cell Sorting System with BD FACSDiva Software (BD Biosciences, Franklin Lake, NJ) on unfixed specimens. Sorted cells were dispensed into a 5,184-nanowell version 1 microchip using the ICELL8 platform (WaferGen Biosystems, Fremont, CA) from which single cells were selected for library preparation and sequencing. Library preparation was carried out using ICell8 single-cell poly-(A)+ transcriptome amplification hands-on workflow (with Triton X-100 for cell lysis), protocol D07-000040-003 rev4. Libraries were generated for a total of 1,134 selected cells, which were then sequenced as 100 base pair single-end reads on an Illumina HiSeq 2500 (Illumina, Inc., San Diego, CA) in three batches (two flow cell lanes per run).
GEO Accession
GSM3391587