organism: Gallus gallus
description: Protocols: Hypothalamus samples were collected from 35 days old chickens. Samples were snap frozen in liquid nitrogen. standard Cobb500 rearing condition as recommended by the supplier. TM: 39.5°C and 65% RH 12h/days from embryonic days E7 to E16 and the rest of the time 37,8°C 55% RH during embryogenesis. No treatment (Control): 37,8°C and 55% RH during embryogenesis. Tissues were ground in a mortar using a pestle both cooled using liquid nitrogen. Ground powder was transferred in 1.5 mL TPX tube containing fixation buffer (1% FA in PBS with 1 X Complete™ protease inhibitors, PBS-C, Roche) for 5 min at room temperature under agitation. The reaction was stop by adding Glycine to 0,125M final. The samples were centrifuged for 5min at 3000g and washed twice using 1 mL ice-cold PBS-C. Cells were then centrifuged 5 min at 5000 g and cellular pellets were resuspended in 300 μL sodium Dodecyl Sulphate (SDS) Lysis Buffer (50 mM Tris-HCl pH 8, 10 mM EDTA pH 8, 1% SDS, 10% glycerol, 1 X Complete™ protease inhibitors). Tubes were vortexed 20 s, incubated 10 min on ice and vortexed 20 s again. Lysed nuclei were sonicated using a Bioruptor (Diagenode, Denville, USA) set to high setting (30 s ON, 30 s OFF; 4 × 5 min). Chromatin immunoprecipitation was performed as described in David et al., 2017 (doi: 10.1186/s12575-017-0059-0). Antibodies batches used: Millipore 07-449#2506493 (H3K27me3); Millipore 07-473#JBC188194 (H3K4me3). Sequencing libraries were prepared using the NEB Next Ultra II DNA Library Prep kit for Illumina (New England Biolabs, Ipswich, USA). Ten ng of H3K27me3 IP DNA, 10 ng of H3K4me3 IP DNA and 100 ng of input DNA were used. Agencourt AMPure XP beads (Beckman Coulter, Brea, USA) were used for the 200 bp size selection of DNA fragments. Libraries concentrations were measured by Qubit and stored at −80 °C until sequencing.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-01-31T17:02:07Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-10-17T11:28:23Z
attributes.tag: External Id
attributes.value: SAMEA4979749
attributes.tag: INSDC center name
attributes.value: BOA, INRA, Universite de Tours
attributes.tag: INSDC first public
attributes.value: 2019-01-31T17:02:07Z
attributes.tag: INSDC last update
attributes.value: 2018-10-17T11:28:23Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7300:C8865_H3K4me3_VTCM64.fastq.gz
attributes.tag: age
attributes.value: 35
attributes.tag: breed
attributes.value: Cobb500
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: chicken
attributes.tag: organism part
attributes.value: hypothalamus
attributes.tag: sample name
attributes.value: E-MTAB-7300:C8865_H3K4me3_VTCM64.fastq.gz
attributes.tag: scientific_name
attributes.value: Gallus gallus