2024-11-19
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114295317
External Id
Tel Aviv university
INSDC center name
2023-08-22T14:16:18Z
INSDC last update
public
INSDC status
ERS16284276
SRA accession
E-MTAB-13275:lung_m2
Submitter Id
ArrayExpress
broker name
not collected
collection date
house mouse
common name
Protocols: Lung tissues were removed following transcardial perfusion and washed in cold D-PBS (Biological Industries, 02-023-1A). Trachea was removed and the tissue was immediately injected with an enzyme mix based on previous work [A cellular census of human lungs identifies novel cell states in health and in asthma] (Dispase II 50 caseinolytic u/ml (Sigma-Aldrich, D4693), Elastase 4.3 u/ml (Worthington Biochem, Worthington, LS002292)), Dnase I 30 μg/ml (Roche), Collagenase A 2 mg/ml (Roche, 10103578001), CaCl2 5 mM) followed by mincing and a brief incubation in a CO2 incubator at 37 ºC. Samples were then transferred to a shaker-incubator for 20 min at 37 ºC, 150 rpm, followed by an immediate addition of 15 ml of neutralization buffer (DMEM (Biological industries, 01-052-1A), 10% FBS (Rhenium, 10270106). Samples were then passed through a 40 um strainer twice and pelleted at 400 G, 5 min at 4 °C. Samples were then treated with RBC cell lysis (Sigma, R7757 as according to the manufacturer’s protocol) and quenched with (10% FBS, D-PBS). Cells were then pelleted at 400 G, 5 min at 4 °C, resuspended in DMEM and counted. Dead cell removal protocol (Miltney, 130-090-101 ) was applied according to manufacturer protocol in cases when cell viability was lower than 75%, and the flowthrough was pelleted at 400 G ,5 min at 4 °C and resuspended in PBS with 0.05% BSA Cells were counted and loaded on the 10X Genomics Chromium instrument for single-cell sequencing, using 3’ V2 chemistry, according to the manufacturer’s protocol. cDNA library preparation was were carried out according to the manufacturer’s protocol.
description
adult
developmental stage
not collected
geographic location (country and/or sea)
m1
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
lung
organism part
Mus musculus
scientific_name
male
sex
C57BL/6
strain
lung_m2
title