Bulk RNA-seq of Cd4 T cells from mice with MC38 tumors
Bulk RNA-seq of Cd4 T cells from mice with MC38 tumors
For conventional CD4+ T and regulatory (GFP+) CD4+ T cell sorting, cells from lymph node digestion were enriched for CD4+ T lymphocytes by negative selection with the EasySep mouse CD4+ T cell Isolation Kit (STEMCELL technologies cat. 19852), cells from tumor digestion were enriched for CD45+ T by positive selection with the EasySep mouse CD45+ positive cell Isolation Kit (STEMCELL technologies cat. 18945). The enriched lymphocytes were subsequently stained and FACS-sorted for bulk RNAseq. Cells were stained for surface antigens (CD8-PE, CD45.2 PE-Dazzle, Thy1.2 Alexa-700, CD4 BUV395). Viable T cells from tumor and dLN were sorted as CD45.2+Thy1.2+CD4+CD8- GFP+ (Regulatory CD4 T) or CD45.2+Thy1.2+CD4+CD8- GFP- (Conventional CD4). Cells were sorted using a BD FACSAria™ Fusion flow cytometer (BD Biosciences, San Jose, CA). 2 to 33 thousand cells were sorted in 300 μL of PBS + 2% FCS and transferred in RLT buffer. RNA was extracted with RNeasy Mini Kit (Qiagen) for RNA sequencing. Total RNA was quantified with Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and quality was assessed using RNA ScreenTape on 4200 TapeStation (Agilent Technologies). Libraries were generated from 2 nanograms of total RNA using Smart-Seq V4 Ultra Low Input RNA Kit (Takara); 150 picograms of cDNA was used to make sequencing libraries by Nextera XT DNA Sample Preparation Kit (Illumina). Libraries were quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the average library size was determined using D1000 ScreenTape on 4200 TapeStation (Agilent Technologies).