GSM8305925: PD-L1 positive D11; Mus musculus; RNA-Seq
Freshly collected lungs were harvested and single cell suspension of immune cells was performed. Cell suspensions were labelled with antibodies and neutrophils were purified using a flow cytometry-based sorting (FACS Melody, BD Biosciences) as live CD45+ SiglecF- CD11b+ Ly6G+ PD-L1+/-. Cells were collected in low binding tubes containing PBS pH 7.2 supplemented with 0.04% BSA (Mg2+/Ca2+ EDTA free). Libraries were performed according to the manufacter's instructions (Chromium Next GEM Single Cell 3' Reagent Kit v3.1 Dual Index). Briefly, Gel Beads-in-emulsion (GEM) were generated by combining barcoded gel beads, partitioning oil and the Master Mix containing neutrophils onto a Chromium NextGEM Chip G. After GEM generation, gel beads were dissolved and partitioned cells were lysed before reverse transcription (RT) to obtain barcoded, full-length cDNA from poly-adenylated mRNA (primers contained an Illumina TruSeq Read 1, a 10x Barcode, a Unique Molecular Identifier UMI and poly(dT) sequence). The pooled barcoded cDNA was then cleaned up with Silane magnetic beads to purify the first-strand cDNA and amplified via PCR to generate sufficient mass for library construction. SPRIselect reagent was used to optimize the cDNA fragment size. During the library construction, Illumina TruSeq Read 2 primer, i7 and i5 sample indexes and paired-end constructs with P5 and P7 sequences were added via End Repair, A-tailing, Adaptor Ligation and PCR. The final step was sequencing using an Illumina NovaSeq 6000.