2025-12-25
ENA first public
ERC000011
ENA-CHECKLIST
Stanford University, Department of Pathology
INSDC center name
public
INSDC status
ERS27210520
SRA accession
E-MTAB-16050:B16-mtD2(+)2
Submitter Id
ArrayExpress
broker name
B16-F0 (CRL-6322)
cell line
melanocyte
cell type
not collected
collection date
house mouse
common name
Protocols: After tumor-immune co-cultures, cells were resuspended in FACS buffer (PBS in 0.5% BSA) and stained with DAPI and CD45. Live tumor cells (TdTomato+CD45-DAPI-) were sorted into Dendra2+ and Dendra2- subpopulations and pelleted immediately. Total RNA was isolated using Qiagen RNEasy Plus isolation kit. 1) mRNA enrichment and purification: Oligo dT Selection (mRNA enrichment): Use oligo dT beads to enrich mRNA with poly A tail. 2) RNA fragment and reverse transcription Fragment the RNA and first-strand cDNA was generated using random N6-primed reverse transcription, followed by a second-strand cDNA synthesis with dUTP instead of dTTP. 3) End repair, add A and adaptor ligation The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; 4) PCR amplification Prior to PCR amplification, the dUTP-marked strand is selectively degraded by Uracil-DNA-Glycosylase(UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing. Many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer; 5) Single strand separation and cyclization Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase; 6) DNA nanoball synthesis
description
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
2
replicate
mitochondria acquired from leukocyte
response to treatment
Mus musculus
scientific_name
B16-mtD2(+)2
title