GSM4561020: NAM_treated TSA CD45+ infiltrating cells; Mus musculus; RNA-Seq
TSA tumors were recovered from 3 mice per group, and dissociated using the Tumor Dissociation Kit and gentleMACS™ Octo Dissociator (Miltenyi Biotec), following manufacturer's instructions. Upon pooling, samples were subjected to magnetic separation of CD45+ cells using magnetic beads and MACS separator (Miltenyi Biotec), as per manufacturer's instructions. Upon testing viability by trypan blue exclusion using a Cellometer AutoT4 cell counter (Nexcelom Bioscience), CD45+ cells were resuspended in PBS containing 0.1% BSA. After CD45+ cells isolation, all procedures were done by the epigenomics core at WCMC. Library preparation for Single Cell 3' RNA-seq v.2, sequencing and post-processing of the raw data was performed at the Epigenomics Core at Weill Cornell Medicine, as follows. Single-cell RNA-seq libraries were prepared according to 10X Genomics specifications (Single Cell 3′ Reagent Kits v2 User Guide PN-120236, 10x Genomics, Pleasanton, CA, USA). Two independent cell suspensions (~60% viable) at a concentration of ~900 cells µL-1 were loaded onto to the 10x Genomics Chromium platform to generate barcoded single-cell GEMs, targeting about 3000 single cells per sample. GEM reverse transcription (53°C for 45 min, 85°C for 5 min; held at 4°C) was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad, Hercules). After reverse transcription, GEMs were broken and single-strand cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, Waltham, MA). cDNA was amplified for 12 cycles (98 °C for 3 min; 98 °C for 15 s, 67 °C for 20 s, 72 °C for 1min) using the C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. Quality of the cDNA was assessed using an Agilent Bioanalyzer 2100 (Santa Clara, CA), obtaining a product of about 1693bp. This cDNA was enzymatically fragmented, end-repaired, A-tailed, subjected to a double-sided size selection with SPRIselect beads (Beckman Coulter, Indianapolis, IN) and ligated to adaptors provided in the kit. A unique sample index for each library was introduced through 13 cycles of PCR amplification using the indexes provided in the kit (98 °C for 45 s; 98 °C for 20 s, 54 °C for 30 s, and 72 °C for 20 s x 14 cycles; 72 °C for 1 min; held at 4 °C). Indexed libraries were subjected a second double-sided size selection, and libraries were then quantified using Qubit fluorometric quantification (Thermo Fisher Scientific, Waltham, MA). The quality was assessed on an Agilent Bioanalyzer 2100, obtaining an average library size of 455bp. Libraries were finally diluted to 2 nM and clustered on a HiSeq2500 high output mode at 10 pM on a pair-end read flow cell and sequenced for 26 cycles on R1 (10X barcode and the UMIs), followed by 8 cycles of I7 Index (sample Index), and 98 bases on R2 (transcript), with a coverage around 100M reads per sample. Primary processing of sequencing images was done using Illumina's Real Time Analysis software (RTA).
GEO Accession
GSM4561020