GSM7035956: VEH+LPS, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Freshly dissected lungs were pooled in 5 ml of FACS buffer-1X PBS, 1% Fetal Bovine Serum (FBS), and 2mM EDTA, homogenized into a single cell suspension, and filtered with 40uM strainer. Next, the single-cell suspension was centrifuged at 1300 rpm for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in 1mL of RBC lysis buffer and placed on ice for 5 minutes, and the single-cell suspension was then treated with 5 ml of FACS buffer and centrifuged at 1300 rpm for 5 min at 4°C. After discarding the supernatant, cells were washed and centrifuged twice more. The cell pellet was resuspended in 1xPBS with 0.04% w/v BSA buffer and filtered. Using the manufacturer's protocol exclusively for gene expression, the single-cell RNA-seq libraries were generated from the samples using the Chromium v2 single-cell 5′ VDJ RNA-seq reagent kit [10x Genomics], 3000 target cells per sample (Vehicle, LPS+Veh, LPS+I3C) were loaded into separate lanes onto the 10x Genomics Chromium Controller to generate the gel bead-in-emulsion (GEM) that has polyA mRNA. Samples were collected and placed on the thermocycler. Now samples are cDNA that contain R1, 10x barcode, unique molecular identifier (UMI) and template switch oligo. Amplified cDNA was cleaned up with Silane DynaBeads. Cleaned cDNA was enzymatically fragmented and the correct size selected utilizing SPRIselect breads from [Beckman Coulter]. The optimized amplicon then ligated with R2 primer sequence, and indexed with both the P5 and P7 primers.