Illumina HiSeq 2500 paired end sequencing; Single-cell ATAC-seq of mouse splenocytes
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
ATAC-Seq
Antigen
ATAC-Seq
Cell type
Cell type Class
Spleen
Cell type
Splenocytes
NA
NA
Attributes by original data submitter
Sample
ENA first public
2018-10-10
ENA last update
2018-05-02
ENA-CHECKLIST
ERC000011
External Id
SAMEA4626653
INSDC center alias
Wellcome Trust Sanger Institute
INSDC center name
Wellcome Trust Sanger Institute
INSDC first public
2018-10-10T17:03:07Z
INSDC last update
2018-05-02T11:14:35Z
INSDC status
public
Submitter Id
E-MTAB-6714:mSp_rep3_081
age
8
broker name
ArrayExpress
cell type
splenocyte
common name
house mouse
genotype
wild type genotype
individual
mouse 1
organism part
spleen
post analysis well quality
pass
sample name
E-MTAB-6714:mSp_rep3_081
sex
male
strain
C57BL/6J
Sequenced DNA Library
library_name
mSp_rep3_081_p
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
The spleen from a C57BL/6Jax mouse was mashed by a 2-ml syringe plunger through a 70 μm cell strainer (Fisher Scientific 10788201) into 30 ml 1X DPBS (ThermoFisher 14190169) supplied with 2 mM EDTA and 0.5% (w/v) BSA (Sigma A9418). Cells were centrifuged down, supernatant was removed, and the cell pellet was briefly vortexed. 5 ml 1X RBC lysis buffer (ThermoFisher 00-4300-54) was used to resuspend the cell pellet, and the cell suspension was vortexed again, and left on bench for 5 minutes to lyse red blood cells. Then 45 ml 1X DPBS was added, and cells were centrifuged down. 30 ml 1X DPBS were used to resuspend the cell pellet. The cell suspension was passed through a Miltenyi 30 μm Pre-Separation Filter (Miltenyi 130-041-407), and the cell number was determined using C-chip counting chamber (VWR DHC-N01). All centrifugations were done at 500 g, 4 °C, 5 minutes. After tagmentation, DAPI was added at a final concentration of 1 ug/ml. Then DAPI positive nuclei were sorted into 384-well plate using BD-INFLUX sorter. The nuclei sorting was performed at the Flow Cytometry facility at the Wellcome Trust Sanger Institute. Nuclei prepration and ATAC-seq procedure are performed simultaneously, i.e. Fast-ATAC protocol was used (Corces et al. Nature Genetics, 48, 1193-1203, 2016). Briefly, 50,000 cells were pelleted down at 500 g, 4 degree for 5 minutes. Then cell pellet was resuspended in 50 ul tagmentation mix containing 33 mM Tris-acetate, pH7.8, 66 mM potassium acetate, 10 mM magnesium acetate, 16% (v/v) dimethylformamide, 0.01% digitonin, 5 ul Tn5 transposes (Nextera kit, Illumina Cat No. FC-121-1030). The tagmentation reaction was put on a thermomixer, with 37 degree for 30 minutes. Then reaction was stopped by adding 50 ul 20 mM EDTA pH 8.0. After tagmentation, DAPI was added at a final concentration of 1 ug/ml. Then DAPI positive nuclei were sorted into 384-well plate using BD-INFLUX sorter. The nuclei sorting was performed at the Flow Cytometry facility at the Wellcome Trust Sanger Institute. DNA was purified first by Qiagen minElute PCR purification kit according to the manufacturer's instruction, then by AmupureXP beads with 0.5X upper cutoff and a 1.2X lower cutoff according to manufacturer's instruction. Libraries were constructed in the Illumina Nextera DNA Library preparation kit.