Design: wnt1-RNAi 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. 20 animals were used per biological replicate. Planarian mucous was removed by washing in 2% L-Cystein (pH 7) for 2. Afterwards, animals were transferred to a Petri dish with CMFH (2.56mM NaH2PO4x2H2O, 14.28mM NaCl, 10.21mM KCl, 9.42mM NaHCO3, 1% BSA, 0.5% Glucose, 15mM HEPES pH 7.3). Planarians were placed in Peltier Cells at 8C to amputate the wound region (the blastema and post-blastema region posterior to the mouth). Samples were transferred to a 1.5ml Eppendorf tube to be dissociated using a solution of liberase/CMFH (1:10) at RT for 10. ATAC-sequencing was carried out as first described in Buenrostro et al (Curr Protoc Mol Biol, 21:29.1-29.9, 2015) and then adapted by Lukoseviciute et al (Dev Cell, 47:608-628, 2018). The solution was transferred to a 50ml falcon tube containing 4ml of Hanks solution (1xHBSS, 10mM HEPES, 0.25% BSA). The eppenford was rinsed with 1ml of Hanks solution to recover more material. The falcon was centrifuged 800g 8, the supernatant was discarded and the pellet was resuspended in 4ml of Hanks solution. The solution was passed through a 40um cell strainer and centrifuged again 8 at 800g. The supernatant was removed and the pellet was resuspended in 1ml of cold PBS 1X and transferred to a new eppendorf. The sample was centrifuged 500g, 10 at 4C, the supernatant was removed and the pellet was resuspended in 50ul of cold 1xPBS. The sample was centrifuged again 500g, 10 at 4C, the supernatant was removed and resuspended in 50ul of cold lysis buffer (5ul of Tris HCl pH 7.4, 1.5ul of MgCl 2 , 10ul of 10% NP40, and 1ul of NaCl 5M, made up to 500ul with nuclease free water). 5ul of the resuspended sample were taken to count the number of nuclei in a Neubauer chamber. The volume corresponding to 50,000 cells was transferred into a new eppendorf. The sample was centrifuged 500g, 10 at 4C and resuspended in transposition reaction (10ul of 2x TD Buffer, 1ul Tn5, 9ul nuclease free water). The reaction was incubated 30 at 37C; 2.4ul EDTA 0.5M were added and incubated at 55C for 30. After that, 1.2ul MgCl 2 was added and the samples were purified using Qiagen Mini Cleanup Kit, according to the manufacturers, and resuspended in 10ul of nuclease free water. The DNA fragments were amplified by carrying out the following PCR reaction: 10ul of transposase DNA, 10ul on nuclease free water, 2.5ul of 25uM adapter 1, 2.5ul of 25uM adapter 2, and 25ul of NEBNext High Fidelity PCR 2X; cycling as follows: 75C for 5, 98C for 30, 16 cycles of 98C for 10, 63C for 30, and 72C for 1, and hold at 4C. The PCR reaction was purified using Qiagen Mini PCR Purification and resuspended in 10ul of nuclease free water. AMPure beads purification SRI1X was carried out; the libraries were quantified using Qubit, 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: ATACseq wnt1-RNAi 12h posterior blastemasLibrary:
Name: WNTBCAT_ATACseq_12h_WNT_rep3
Instrument: Illumina HiSeq 4000
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Runs:
1 run, 9.1M spots, 894.4M bases, 359.1Mb