GSM8179700: ColonLP_myeloid_KO_aPD1_2; Mus musculus; RNA-Seq
For scRNAseq: Using the Chromium Single Cell 3′ Reagent Kit v2 or v3User Guide, single cell suspensions were partitioned into nanoliter droplets called Gel-bead-in-Emulsions (GEMs) to achieve single cell resolution. The cDNA generated within each individual GEM is tagged with a common 16nt 10x barcode and a 12nt unique molecular barcode during the RT reaction. Purified cDNA was amplified for 11 cycles before being purified using SPRIselect beads. Samples were then run on a Agilent Bioanalyzer to determine cDNA concentration. 10uL of purified cDNA was used to generate the Illumina library for sequencing. For Bulk RNA seq: total tissue RNA was extracted using RNA easy mini kit. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. For scRNAseq: For sample preparation on the 10x Genomics platform, the Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 16 rxns (PN-1000075), Chromium Chip B Single Cell Kit, 48 rxns (PN-1000073) and Chromium i7 Multiplex Kit (PN-120262) were used. The single cell libraries were then sequenced on the Illumina NovaSeq 6000 S4 200 cycle flow cell generating 28x98 reads. A median sequencing depth of 50,000 reads/cell was targeted for each sample. For bulk RNAseq: Library preparation was performed with 5 to 10ug of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags.