GSM3323499: TANs single-cell RNA-seq; Mus musculus; RNA-Seq
CD45+CD11B+LY6G+ neutrophils were flow sorted and pooled to prepare TAN single cell population (n = 9 tumors from 2 SNL mice) and PBN population (n = blood from 2 SNL mice). Without any freezing or fixation steps, the single cell populations were immediately processed. Cell suspensions were partitioned into an emulsion of nanoliter-sized droplets using a 10X Genomics Chromium Single Cell Controller and RNA sequencing libraries were constructed using the Chromium Single Cell 3' Reagent Kit v2 (10X Genomics Cat#PN-120237). Briefly, droplets contained individual cells, reverse transcription reagents and a gel bead loaded with poly(dT) primers that include a 16 base cell barcode and a 10 base unique molecular index. Lysis of the cells and gel bead enables priming and reverse transcription of poly-A RNA to generate barcoded cDNA molecules. Libraries were constructed by End Repair, A-Tailing, Adapter Ligation and PCR amplification of the cDNA molecules. Purified cDNA libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (Agilent Cat#5067-5582 and Cat#5067-5583). The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (Kapa Biosystems Cat#KK4824). HiSeq 125 Cycle Paired-End Sequencing v4: Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 paired end flow cell using an Illumina cBot.  Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq PE Cluster Kit v4-cBot (PE-401-4001).  Following transfer of the flowcell to an Illumina HiSeq 2500 instrument (HCS v2.2.38 and RTA v1.18.61), a 125 cycle paired-end sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC-401-4003).
GEO Accession
GSM3323499