2024-12-06
ENA first public
ERC000011
ENA-CHECKLIST
Genentech
INSDC center name
public
INSDC status
ERS21395607
SRA accession
E-MTAB-14581:LN_T_OE_12
Submitter Id
ArrayExpress
broker name
T cell
cell_type
not collected
collection date
house mouse
common name
Protocols: 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).
description
adult
developmental stage
not collected
geographic location (country and/or sea)
Animal_12
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
lymph node
organism part
Mus musculus
scientific_name
female
sex
C57BL/6J
strain
LN_T_OE_12
title