2024-04-01
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA115098379
External Id
Genentech
INSDC center name
2024-01-11T21:25:33Z
INSDC last update
public
INSDC status
ERS17733126
SRA accession
E-MTAB-13698:Bolus
Submitter Id
ArrayExpress
broker name
myeloid cell
cell_type
not collected
collection date
house mouse
common name
Protocols: Mice were treated with an MPS or bolus vaccine (containing GM-CSF, CpG, and OVA) and draining LNs were collected on day 21/20 (MPS and bolus) and compared to naïve controls. LNs were digested in RPMI-1640 (Corning) containing 0.8mg/mL Dispase II, 0.2mg/mL Collagenase P, and 0.1mg/mL DNase I (all Roche, procured from Sigma) until no visible LN pieces remained and passed through a 70μm filter. Samples were incubated in TruStain FcX (Fc block, 1:200, BioLegend) prior to incubation with staining antibodies (PE/Cy7 anti-mouse CD45, BV711 anti-mouse CD3, BV421 anti-mouse CD19, FITC anti-mouse CD31, and APC anti-mouse podoplanin, all BioLegend) and one Hashtag antibody per replicate using BioLegend TotalSeq-B03 (catalog numbers 155831, 155833, 155835, 155837, 155839; barcode sequences ACCCACCAGTAAGAC (-B0301), GGTCGAGAGCATTCA (-B0302), CTTGCCGCATGTCAT (-B0303), AAAGCATTCTTCACG (-B0304), CTTTGTCTTTGTGAG (-B0305), all diluted 1:100 in FACS buffer). Cell viability was assessed through eBioscience Fixable Viability Dye eFluor 780 (ThermoFisher). Cells were gently washed and resuspended in PBS + 2% FBS prior to sorting. Myeloid (viable, CD45+CD3-CD19-) and stromal (viable, CD45- FSC-Ahi) cells were enriched using a BD FACS Aria cell sorter into RPMI + 30% FBS on ice. Cells were counted and resuspended at an adequate concentration for loading. Sorted cells were loaded onto a 10x Chromium Chip using reagents from the Chromium Single Cell 3’ Library Kit (10x Genomics) according to the manufacturer’s protocol. Amplified cDNA was used for 3’ RNA-seq library generation, RNA-seq libraries were prepared following the manufacturer’s user guide (10x Genomics).
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
lymph node
organism part
Mus musculus
scientific_name
female
sex
C57BL6J
strain
Bolus
title