GSM4662024: YueLab-H3K9me2-Testis-rep1; Danio rerio; ChIP-Seq
RNA-seq: For each RNA-seq experiment, the same tissues combined from at least two Tuebingen fish were used as one replicate. For embryonic trunk, ten 1-dpf fish were dechorionated with pronase and trunk were cut off for RNA-seq. For embryonic neuron, green cells from Tg(Huc:Kaede) cells were sorted by FACS and approxinately 20,000 cells were used for one replicate. The tissue RNA was extracted from Trizol® according to the protocol (Invitrogen). RNA-seq: The cDNA libraries were performed using SureSelect Strand Specific RNA Library Preparation Kit (Agilent) according to the manufacturer's protocol. Briefly, polyA RNA was purified from 1000 ng of total RNA using oligo (dT) beads (Invitrogen). Extracted RNA was first fragmented, then followed by reverse transcription, end repair, adenylation, adaptor ligation and subsequent PCR amplification. The final product was checked by size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent) and Kapa Library Quantification Kit (Kapa Biosystems) and then followed by pair-end 2X 50 bp high-throughput sequencing using HiSeq 2500 (Illumina). ChIP-seq: All the tissues were ground in the liquid nitrogen and fixed by 1% formaldehyde at room temperature for 15 min. 0.2 M glycine was added and incubated at room temperature for 5 min to quench the fixation. Fixed tissues were then washed once by cold 1X PBS. Tissue pellet was resuspended and incubated on ice for 10 min in 100 µL of ChIP-seq lysis buffer (20 mM Tris-HCl, pH 8.0, 1% SDS, 50 mM EDTA, 1X proteinase inhibitor cocktail). Next, 900 µL cold 1X TE buffer was added to dilute SDS concentration and the nuclei suspension was sonicated using Covaris E220 with the following parameters: 140 W, duty factor 5, 200 per burst. Sonication time is variable, depending on tissue types. To check the chromatin fragmentation size, 20 µL of input chromatin was reverse crosslinked in elution buffer (20 mM Tris-HCl, pH 8.0, 1% SDS, 1 mM EDTA) at 65 °C overnight, treated with Rnase A and proteinase K and purified by phenol-chloroform extraction. Input DNA was then loaded on a Lonza flash gel to make sure that the majority of DNA size distributes between 100-300 bp. To prepare antibody-beads complex, 3 µg of histone H3K27ac antibody (Active Motif, 39133) or H3K4me3 antibody (EMD Millipore, 07-473) was mix with 12 µL M-280 sheep anti-rabbit IgG Dynabeads (ThermoFisher, 11203D) in 150 µL of 5 mg/mL BSA/1X PBS buffer, with rotation at 4 °C for 3 h. After incubation, antibody-beads complex was washed once with BSA/1X PBS buffer. About 200 µg chromatin was used per IP. Equal volume of master mix (1X TE, 2% Triton X-100, 0.2% sodium deoxycholate, 2X proteinase inhibitor cocktail) was mixed with 200 µg chromatin and then incubated with antibody-beads complex overnight with rotation. The next morning, the beads were washed 5 times with cold RIPA wash buffer (20 mM Tris-HCl, pH 8.0, 1% NP-40, 0.7% sodium deoxycholate, 500 mM LiCl, 1 mM EDTA, 1X proteinase inhibitor cocktail). Then the chromatin bound on the beads was eluted by 150 µL of elution buffer at 65 °C for 30 min. ChIP-seq: To prepare the library, eluted chromatin was reverse crosslinked and purified by phenol-chloroform extraction. Then DNA was end-repaired by END-IT DNA end-repair kit (Epicentre, ER81050) according to kit's protocol, added A using Klenow  fragment (3'->5' exo-) (NEB, M0212S), ligated with Illumina TruSeq adaptor (Illumina, FC-121-3001) and subsequently amplified by PCR (Roche, kk2601). The quality and quantity of all the libraries were checked using BioAnalyzer High Sensitivity DNA Kit (Agilent). The libraries were sequenced on Illumina HiSeq 2500 with reads length of 2X 50 bp or 2X 100 bp. ATAC-seq: Tuebingen adult tissues were ground in liquid nitrogen,resuspended in 1 mL of lysis buffer (1X PBS, 0.2% NP-40, 5% BSA, 1 mM DTT, protease inhibitors), followed by incubation at  4 °C for 10 min on an overhead shaker. After that, lysate was filtered through 40 m cell strainer and nuclei were collected at 1000X g for 5 min. ATAC-seq: Tagmentation was performed immediately according to ATAC-seq protocol reported previously(Buenrostro et al., 2013) Hi-C: For brain tissue, two adult Tuebingen brains were slightly ground into small granules in liquid nitrogen and resuspended in 1 mL of cold hypotonic buffer (20 mM Tris-HCl, pH 8.0, 10 mM NaCl, 20 mM EDTA). Granular brains were then dounce homogenized with loose pestle for 20 times. The top of the homogenate was carefully transferred into a new tube and fixed with 2% formaldehyde at room temperature for 10 min. 0.2 M of glycine was added to stop fixation. For muscle tissue, 60 mg of Tuebingen muscle was first chopped into small pieces and digested by 0.25% trypsin at room temperature for 30 min. After neutralization with FBS, muscle cells were resuspended in cold 1X PBS and fixed with 2% of formaldehyde at room temperature for 10 min. 0.2 M glycine was then added to stop fixation. Hi-C: Hi-C experiments on adult zebrafish brain and muscle tissues were performed according to previously published protocol(Rao et al., 2014). The final product was checked by size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent) and Kapa Library Quantification Kit (Kapa Biosystems) and then followed by pair-end 2X 100 bp high-throughput sequencing using HiSeq 2500  or HiSeq 4000(Illumina). WGBS: Tübingen adult tissues were dissected, and the genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, 69504). Then 1 µg of genomic DNA of each adult tissue was subjected to bisulfite conversion using EZ DNA Methylation-Gold Kits WGBS: The final libraries were prepared using the Accel-NGS® Methyl-Seq DNA Library Kit. scATAC-seq: Single-cell ATAC-seq (scATAC-seq) was performed in one female brain and one male brain, respectively, on the 10X Genomics platform. To isolate nuclei, freshly dissected single brain was transferred to 1 mL NbActiv1 medium (BrainBits, NbActiv1 500) with a wide-bore pipette tip to break the tissue into small pieces. Then the tissue was broken up completely using regular-bore pipette tips followed by filtering through a 30 µm cell strainer. The isolated cells were spun down at 500 g for 5 min at 4 °C and then lysed in 100 µL chilled 0.1X Lysis Buffer (10 mM Tris HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% BSA,0.01% Tween-20, 0.01% NP40 and 0.001% digitonin) on ice for 5 min. Then 1 mL chilled Wash Buffer (10 mM Tris HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20) was added to the lysed cells and cells were spun down at 500 g for 5 min at 4 °C. At last, 300 µL chilled Diluted Nuclei Buffer (10x Genomics, PN 2000153/2000207) was added to resuspend the nuclei. The nuclei were filtered again using a 30 µm cell strainer before cell counting scATAC-seq: Around 12,000 nuclei were used for one Tn5 tagmentation reaction, and the scATAC-seq library was prepared and sequenced exactly according to the company's user guide.
GEO Accession
GSM4662024