GSM9207212: Sample3-GEX; Mus musculus; RNA-Seq
Lung samples were homogenized in TRIzol (Invitrogen #15596026) followed by adding 200 μl Chloroform. Samples were well-mixed and centrifuged at 12,900 g for 15 min. The equal volume of 2-propanol was mixed into the supernatant in a new tube and the samples were precipitated on ice for 10 min followed by 15 min centrifuge at 12,900 g. The supernatant was discarded. The precipitated pellets were washed by 75 % ethanol for twice and air-dried for 5 min. RNA samples were resuspended in nuclease-free water and stored at -80 °C. Hashtag antibodies (TotalSeqTM-C0301 to C315 anti-mouse Hashtag 1 to 15 Antibody, Biolegend #155861 to #155889) were added into the staining cocktail. Macrophages (Live/CD45+/IV-/B220-/CD90.2- /Ly6G-/Ly6C-/MertK+/CD64+) were sorted from these samples and pooled together for generating library. The library was prepared based on Chromium Next GEM Single Cell 5' Reagent Kits v2 (10X Genomics, document number: CG000220 Rev F). The sequencing was done at Center for Human Immunology/NIAID with NextSeq 1000/2000 P2 XLEAP-SBS Reagent Kit (Illumina #20100987). The parameters were Read 1/Index 1/Index 2/Read 2: 27 / 10 / 10 / 91 cycles.