2025-07-30
ENA first public
ERC000011
ENA-CHECKLIST
Cambridge Institute for Medical Research
INSDC center name
public
INSDC status
ERS21130238
SRA accession
E-MTAB-14519:Sample 4
Submitter Id
9
week
age
ArrayExpress
broker name
CD8-positive, alpha-beta T cell
cell_type
not collected
collection date
house mouse
common name
Protocols: Cells were harvested by centrifugation into a cell pellet, and lysed with 350 μL RLT lysis buffer supplemented with 1% v/v beta-mercaptoethanol. Spleens were dissected post-mortem under sterile conditions. Effector OT-I CTLs were differentiated and cultured from murine splenocytes as follows. Naïve OT-I splenocytes were stimulated with 10 nM ovalbumin peptide, SIINFEKL (OVA) for 3 days in complete mouse T cell media: RPMI 1640 medium with 10% FBS, 50 μM beta-mercaptoethanol, 10 U/ml recombinant murine IL-2, 2 mM L-Glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were pelleted and resuspended in fresh media from 3 days after stimulation, expanded in CTL media and nucleofected on day 5 after stimulation with CRISPR/Cas9 RNPs targeting Cdc42 or non-targeting control. Cells were harvested 72 hours after nucleofection. OT-I CTLs from six mice were nucleofected with CRISPR/Cas9 RNPs on day 5 post-stimulation. 72 h later, RNA was extracted from two million CTLs. Lysates were passed through a Qiashredder and frozen. RNA was extracted using the RNEasy Mini Kit, according to the manufacturer’s protocol with on-column DNA digestion with RNase-free DNase I. RNA quality was verified by Bioanalyzer 2100 and quantity was determined using a Qubit Fluorometer (version 4). Libraries were prepared for sequencing with the TruSeq Stranded mRNA kit (Illumina).
description
adult
developmental stage
NT
genotype
not collected
geographic location (country and/or sea)
LOOR8.2d
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
spleen
organism part
4
replicate
Mus musculus
scientific_name
male
sex
C57BL/6
strain
Sample 4
title