Design: Wild type animals were amputated posterior to mouth; collection of wound samples (blastema and post-blastema region) posterior to trunk fragments was performed at 12h of regeneration after amputation. A total of 2000 anterior and posterior blastemas were used; groups of 100 blastemas were done at one time. Planarians were placed in Peltier Cells at 8C to cut the wound region. Wounds were transferred to a Petri dish containing 1M MgCl2 solution, for 1530 rocking at room temperature (RT). PBS 1X was added to remove salts. Blastemas were fixed with formaldehyde 1.85% for 15 rocking at RT. Glycine was added to obtain a final concentration of 0.125M to quench formaldehyde, for 5 at RT, rocking. Then, blastemas were washed 3X with cold PBS 1X, PBS excess was removed and samples were stored at 80C. ChIPmentation was carried out as described in Schmidl et al (Nat. Methods, 12:963965, 2015). Samples were homogenized in 5ml of cell lysis buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 0.3% NP-40, Complete 1X); they were centrifuged at 3500rpm, 5 at 4C, the supernatant was discarded and the pellet resuspended in 660ul Nuclear lysis buffer (50mM Tris-HCl pH 7.5, 10mM EDTA, 1% SDS, Complete 1X) diluted by adding 1.34ml of ChIP Dilution buffer (16.7mM Tris-HCl pH 7.5, 1.2mM EDTA, 167mM NaCl, 0.01% SDS, 1.1% Triton X-100). Samples were split in 1ml aliquots and sonicated in a Covaris M220 with settings: duty factor=10%, PIP=75W, 100=cycles/burst, time=10. Samples were centrifuged 5, max speed at 4C and the supernatant were collected. 20ul were kept as an input control. 5/3x200ul of sonicated chromatin were incubated with 2ul of H3K27ac (Abcam ab4729) at 4C, overnight rotating. Samples were incubated with 50ul of Dynabeads protein G resuspended in ChIP Dilution Buffer 1h at 4C. Beads were washed twice in Wash Buffer 1 (20mM Tris-HCl pH 7.5, 2mM EDTA, 150mM NaCl, 1% SDS, 1% Triton X-100), twice in Wash Buffer 2 (20mM Tris-HCl pH 7.5, 2mM EDTA, 500mM NaCl, 0.1% SDS, 1% Triton X-100), twice in Wash Buffer 3 (10mM Tris-HCl pH 7.5, 1mM EDTA, 250mM LiCl, 1% NP-40, Na-deoxycholate 1%), and twice in 10mM Tris-HCl pH 8. Beads were resuspended in TAGmentation reaction mix (10 mM Tris-HCl pH 8.0, 5 mM MgCl 2, 10% w/v dimethylformamide), 1ul of Tn5 was added and incubated for 1. Beads were then washed twice in Wash Buffer 1 and once in TE 1X. The samples were eluted 15 in 100ul of Elution Buffer (50mM NaHCO3 pH 8.8, 1% SDS) and 180ul of Elution Buffer was added to the input sample. 10ul of 4M NaCl and 0.5ul of 10mg/ml proteinase K were added to the ChIP and input sample and it was incubated 4-6h at 65C. DNA was purified using Minelute columns (Qiagen). The library was prepared by PCR: 19ul of DNA, 1ul of 25uM of adapter1 and 2, 25ul of NEBNext High-Fidelity 2X PCR Master Mix (NEB) and 4ul of water which was cycled 98C for 30, 98C for 10, 63C for 30, 72C for 30, and 72C for 5; the number of cycles was empirically determined. The libraries were purified using Minelute columns (Qiagen) and were sequenced on a HiSeq 4000 (Illumina) with a read length of 2x50bp.
Submitted by: Universitat de Barcelona
Study:
Wnt/beta-catenin signalling is required for pole-specific chromatin remodeling during planarian regenerationshow Abstracthide AbstractFor successful regeneration, the identity of the missing tissue must be specified according to the pre-existing tissue. Planarians are ideal for the study of the mechanisms underlying this process because the same field of cells can regrow a head or a tail according to the missing body part. After amputation, the differential activation of the WNT/betacatenin signal specifies anterior versus posterior identity. Initially, both wnt1 and notum (Wnt inhibitor) are expressed in all wounds, but 48 hours later they are restricted to posterior or anterior facing wounds, respectively, by an unknown mechanism. Here we show that 12 hours after amputation the chromatin accessibility of cells in the wound region changes according to the polarity of the pre-existing tissue in a WNT/betacatenin-dependent manner. Genomic analyses suggest that homeobox transcription factors and chromatin-remodeling proteins are direct WNT/\betacat targets, which trigger the expression of posterior effectors. Finally, we identified FoxG as a wnt1 up-stream regulator, probably via binding to its first intron enhancer.
Sample:
Wnt/Bcat signalling chromatin remodeling planarian regeneration: H3K27ac ChIP-seq wt 12h posterior blastemasLibrary:
Name: WNTBCAT_CHIPseq_POST_rep1
Instrument: Illumina HiSeq 4000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Runs:
1 run, 13.4M spots, 1.3G bases, 503.2Mb