2025-07-25
ENA first public
ERC000011
ENA-CHECKLIST
IRB Barcelona, BIST
INSDC center name
public
INSDC status
ERS20766271
SRA accession
E-MTAB-14317:WT
Submitter Id
3
month
age
ArrayExpress
broker name
CD45-positive cells
cell_type
not collected
collection date
house mouse
common name
Protocols: Lungs were collected and were finely minced with a scalpel. The tissue was enzymatically digested in 10 mL of protein-free DMEM containing 1 mg/mL Collagenase A (Roche, #10103586001), 0.2 mg/mL Dispase II (Sigma, #D4693) and 0.2 mg/mL DNAse I (Roche, #10104159001), during 25 min at 37°C with rotation. The enzymatic reaction was quenched by the addition of 30 mL of ice-cold 10% heat-inactivated fetal bovine serum DMEM (10% HI-FBS DMEM). Cell suspension was filtered through a 70 μm cell strainer and the remaining pieces of tissue were smashed against the filter with a 1 mL syringe plunge. The filter was washed with 10 mL of ice-cold 10% HI-FBS DMEM and the cells were pelleted at 300xg for 5 min at 4°C. Erythrocytes were lysed in Red Cell Lysis Buffer during 4 min at RT and the remaining cells were immediately washed with ice-cold 10% HI-FBS DMEM. After filtration through a 70 μm cell strainer and centrifugation, cells were resuspended in ice-cold PBS. Samples were mixed before staining. We used a pool of four mice for each genotype, 1 replicate of each genotype. Prior to surface staining with the conjugated antibodies, cells were incubated 20 min at 4°C with anti-CD16/CD32 to block Fc receptor with Live/Dead Fixable cell dead stain kit, depending on the panel. Cells were then centrifuged and then stained with anti-CD45 BV510, Clone 30F11 (BD-Bioscience) during 30 min at 4°C in the dark. Cells were washed once with FACS buffer and resuspended in FACS buffer. We sorted 200.000 cells of CD45+ by sample using an Aria Sorter (BD), and cells were collected in PBS containing 0.04% of BSA. cell concentration of each sample was adjusted to approximately 1,000 cells/μl for the libraries preparation. the cell concentration of each sample was adjusted to approximately 1,000 cells/μl. Cell partition into GEMs was performed using Chip G and the 3’ v3.1 chemistry (10X Genomics). Barcoded cDNA was amplified for eleven cycles, quantified, and quality controlled on the Bioanalyzer 2100 using a high sensitivity DNA assay (Agilent). Libraries were generated using only 10 μl (25%) of the total cDNA (40 μl), strictly following the manufacturer instructions. 13 amplification cycles were performed for each library. Purified libraries were quality controlled on the Bioanalyzer 2100 and quantified. A 20 nM equimolar pool was prepared and a final qPCR validated libraries before cluster generation.
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
pool of four mice per genotype
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
lung
organism part
Mus musculus
scientific_name
female
sex
C57BL/6J
strain
WT
title