2024-12-05
ENA first public
ERC000011
ENA-CHECKLIST
Genentech
INSDC center name
public
INSDC status
ERS22058385
SRA accession
E-MTAB-14601:Anti_LN_1
Submitter Id
ArrayExpress
broker name
T cell
cell_type
not collected
collection date
house mouse
common name
Protocols: For M86-specific CD8+ T cell sorting (for TCR/scRNA-seq), cells from tumor and lymph node digestion were enriched for CD8+ T lymphocytes by negative selection with the EasySep mouse CD8+ T cell Isolation Kit (STEMCELL technologies cat. 19853). The enriched lymphocytes were subsequently stained and FACS-sorted for scRNAseq. Cells were labelled with PE-conjugated and barcoded (total-seq C0952, BioLegend) M86 tetramer for 20 min at room temperature, then stained for surface antigens (CD8-FITC, CD45.2 PE-Dazzle, Thy1.2 Alexa-700, CD4 BUV395) plus one of the following Hashtag antibodies (one for each replicate, 5 μg/ml): TotalSeqTM-C0301 , -C0302, C0303 , -C0304, -C0305, -C0306, -C0307, -C0308, -C0309, -C0310. Viable T cells from tumor and dLN were sorted as CD45.2+Thy1.2+CD4−CD8+ Tetramer+. Cells were sorted using a BD FACSAriaTM Fusion flow cytometer (BD Biosciences, San Jose, CA). Two to 50 thousand cells were sorted in 300 μL of PBS + 2% FCS and kept at 4 °C. Cells were then counted and resuspended at an adequate concentration for loading into the 10X chip. Gene expression and TCR libraries were generated using the Chromium Single Cell 5′ Library and V(D)J Reagent Kit (10X Genomics) according to manufacturer’s recommendations. 20,000 cells per sample were loaded into each channel of the Chromium Chip, and recommendations were followed assuming targeted cell recovery of 2,000–10,000 cells. 10X Geomics single-cell gene expression libraries were quantified with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and profiled using the Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies).
description
adult
developmental stage
LIB5440301_SAM24399307
expression file prefix
not collected
geographic location (country and/or sea)
LIB5440313_SAM24399307
hto file prefix
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
lymph node
organism part
Mus musculus
scientific_name
female
sex
C57BL/6J
strain
Anti_LN_1
title
LIB5440302_SAM24399307
vdj file prefix