Antigen

Antigen Class

No description

Antigen

NA

Cell type

Cell type Class

Kidney

Cell type

Kidney

MeSH Description

Body organ that filters blood for the secretion of URINE and that regulates ion concentrations.

Sample

ENA first public

2019-01-20

ENA last update

2018-01-25

ENA-CHECKLIST

ERC000011

External Id

SAMEA104548800

INSDC center alias

Prof. Daniel Constam' s group (UPCDA) Ecole Polytechnique Federale de Lausanne (EPFL) SV ISREC, Lausanne, Switzerland.

INSDC center name

Prof. Daniel Constam' s group (UPCDA) Ecole Polytechnique Federale de Lausanne (EPFL) SV ISREC, Lausanne, Switzerland.

INSDC first public

2019-01-20T17:02:07Z

INSDC last update

2018-01-25T13:45:03Z

INSDC status

public

Submitter Id

E-MTAB-6424:6_H3K4me1_KO_male

age

2

broker name

ArrayExpress

common name

house mouse

diet

2 days post natal mice : mother milk ; adult (mother) mice : Breeding diet 3242 from KLIBA NAFAG, PROVIMI KLIBA AG

disease

polycystic kidney disease

genotype

Bicc1 homozygous knock out

organism part

kidney

phenotype

cystic kidney

sample name

E-MTAB-6424:6_H3K4me1_KO_male

sex

male

strain

C57BL/6

Sequenced DNA Library

library_name

6_H3K4me1_KO_male_s

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Two days post-natal Bicc1 WT and KO mice were sacrificed by decapitation. For ChIPseq experiment one kidney per animal was collected. Whole kidneys were incubated in 1% formaldehyde methanol-free (Pierce™ 16% Formaldehyde (w/v), Catalog number: 28906) for 15' just after mice dissection. Fixation was stopped with 3 washes in PBS 1X on ice and stored at -80°C. The contralateral kidneys of the same animals were collected and processed according to the RNAseq protocol. Fixed and frozen whole kidneys (14 kidneys) were thawed on ice. Pools of two kidneys belonging to two different animals, divided per gender and genotype, were homogenized mechanically in RIPA buffer for 1min using a pellet pestle motor cordless on ice. The homogenized samples were sonicated in RIPA buffer for 20' (30'' ON, 30'' OFF, HIGH) in 15ml tube using the Bioruptor® sonication device (Diagenode). The sonicated samples were mixed together according to the gender and genotype and they were used for the Chromatin immuno-precipitation protocol. Only for KO female, the pool of sonicated samples derived from two instead of four different kidneys, due to KO female sample limitation. ChIP experiment was performed using magnetic beads (Pierce Protein A/G Magnetic Beads, Thermo Scientific) and antibodies for the transcription factor HNF4A (Ab41898, Abcam), and for histone modifications H3K27ac (Ab4729, Abcam) and H3K4me1 (Ab8895, Abcam). A fraction of the total sheared chromatin per each condition was kept as input sample. The respective input was used as negative control of HNF4A ChIPs and the histone modifications ChIPs for each condition (genotype and sex). The sonicated chromatin samples were incubated with the above-mentioned antibodies and protein A/G beads over night by constant rotating at 4°C. The ChIP samples were washed six times for 10' at 4°C per each of the following four different buffers: RIPA buffer, RIPA buffer/500mM NaCl buffer, LiCl wash buffer, TE buffer. The samples were incubated in the Elution Buffer at 65°C over-night to anti-crosslink the chromatin. After RNaseA and ProteinaseK treatment, the DNA was purified using MinElute PCR Purification Kit - QIAGEN (Cat. No. 28004) and it was stored at -20°C. Multiplexed libraries were prepared using 5ng of DNA and barcoded adapters for each sample, following TruSeq ChIP Library Preparation kit protocol (Illumina, USA).

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

19377432

Reads aligned (%)

99.2

Duplicates removed (%)

6.0

Number of peaks

221 (qval < 1E-05)

mm9

Number of total reads

19377432

Reads aligned (%)

99.1

Duplicates removed (%)

6.1

Number of peaks

185 (qval < 1E-05)