title: 4_HNF4A_WT_male
organism: Mus musculus
description: Protocols: 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).
attributes.tag: ENA first public
attributes.value: 2019-01-20
attributes.tag: ENA last update
attributes.value: 2018-01-25
attributes.tag: External Id
attributes.value: SAMEA104548809
attributes.tag: INSDC center alias
attributes.value: Prof. Daniel Constam' s group (UPCDA) Ecole Polytechnique Federale de Lausanne (EPFL) SV ISREC, Lausanne, Switzerland.
attributes.tag: INSDC center name
attributes.value: Prof. Daniel Constam' s group (UPCDA) Ecole Polytechnique Federale de Lausanne (EPFL) SV ISREC, Lausanne, Switzerland.
attributes.tag: INSDC first public
attributes.value: 2019-01-20T17:02:07Z
attributes.tag: INSDC last update
attributes.value: 2018-01-25T13:45:03Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6424:4_HNF4A_WT_male
attributes.tag: age
attributes.value: 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: diet
attributes.value: 2 days post natal mice : mother milk ; adult (mother) mice : Breeding diet 3242 from KLIBA NAFAG, PROVIMI KLIBA AG
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: kidney
attributes.tag: phenotype
attributes.value: wild type
attributes.tag: sample name
attributes.value: E-MTAB-6424:4_HNF4A_WT_male
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6