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| Status |
Public on Jan 05, 2022 |
| Title |
H3K9me3 IP, Frog eye, Optic nerve crush, Unoperated eye, Replicate 1 |
| Sample type |
SRA |
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| Source name |
6 unoperated frog eyes, 11 days after contralateral orbital optic nerve crush
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| Organism |
Xenopus laevis |
| Characteristics |
strain: albino, Xenopus Express
age: 1-3 mos. postmetamorphic juvenile frog
surgery: optic nerve crush, unoperated eye
Sex: not determined
antibody: H3K9me3
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| Treatment protocol |
Unilateral orbital optic nerve crushes (Zhao and Szaro 1994, doi: 10.1002/cne.903430112) and complete spinal cord transections (Gibbs and Szaro 2018, doi: 10.1101/pdb.prot101030) were performed on fully anesthetized animals (MS222).
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| Growth protocol |
All animals used in this study were purchased from Xenopus Express, Inc (Brooksville, FL). Prior to surgery, they were acclimated to the lab for at least one week, under a 12L:12D photoperiod, in tanks of condition water at 22C, and fed every other day.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5hmC MeDIP-Seq: Eyes for optic nerve crush studies were rapidly dissected and then homogenized immediately, pooling six eyes for each biological replicate.Total DNA, along with RNA, was isolated using the RNA/DNA/Protein Purification kit from Norgen Biotek Corp. with DNase I treatment, following manufactuer's protocols. For histone ChIP-Seq: Eyes and hindbrain were frozen on dry ice immediately upon dissection. Dissected tissues were frozen immediately on dry ice and stored at 80°C for later processing. Tissues were thawed while homogenizing them in PBS containing protease inhibitor cocktail, in 2-ml Pyrex glass homogenizers. After low-speed centrifugation (1,300 rpm for 5 min at 4°C), pellets were resuspended and cross-linked in PBS with 1% formaldehyde for 10 min at room temperature. Cross-linking reactions were quenched with glycine. Samples were subsequently prepared for lysis and sonication in a Diagenode Standard Bioruptor (Chromatin Shearing and Optimization kit, Diagenode #C01020010). Sonication was performed at the high-power setting for 30 cycles of 30 seconds on and 30 seconds off. This was repeated until the sheared chromatin was 150-350 bp in size, as verified by gel electrophoresis. Immunoprecipitation was done using 5 μg of antibody and 50 μl of DiaMag protein A-coated magnetic beads.
For histone ChIP-seq: Samples for ChIP-seq were prepared using the Manual iDEAL ChIP-seq kit for histones (Diagenode #C1010051) according to the manufacturer’s protocols, and the following antibodies: H3K4me3 (Abcam ab8580; lot GR3201240-1), H3K27ac (Abcam ab4729; lot GR3205523-1), H3K27me3 (Abcam MsMAb ab6002; lot GR275911-8), H3K9me3 (Abcam RbPAb ab8898; lots GR3176466-4/GR250850-1). Samples were subsequently prepared for lysis and sonication in a Diagenode Standard Bioruptor (Chromatin Shearing and Optimization kit, Diagenode #C01020010). Sonication was performed at the high-power setting for 30 cycles of 30 seconds on and 30 seconds off. This was repeated until the sheared chromatin was 150-350 bp in size, as verified by gel electrophoresis. Immunoprecipitation was done using 5 μg of antibody and 50 μl of DiaMag protein A-coated magnetic beads. Barcoded DNA libraries were prepared from the immunoprecipitated DNA, as well as from equal amounts of pre-IP DNA from sheared chromatin, using the NEBNext Ultra II DNA Library Prep kit for Illumina sequencing. Sequencing (Illumina NextSeq500) was performed at the University at Albany Center for Functional Genomics core facility (75 bp, single end reads). 5hmC MeDIP: Genomic DNA (1 μg each) was sonicated for 14 cycles of 15 seconds on and 90 seconds off, using a Diagenode Standard Bioruptor to produce fragments of 200--600 bp in length, which was verified by gel electrophoresis. The sheared DNA was then subjected to 5hmC-IP using the kit’s 5hmC antibody and its non-specific IgG control antibody, overnight at 4°C, according to the manufacturer's instructions (hMeDIP Kit, Diagenode #C02010031, mouse monoclonal mAb). The immunoprecipitated DNA was washed and released from the antibody complex by proteinase-K digestion and resuspended in 100 μl of DNA-IP buffer (DIB) provided by the kit. Library preparation and sequencing was then performed as for histone ChIP-Seq.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
For histone ChIP-Seq and 5hmC DIP-Seq: Adapter sequences and barcodes were removed using Trimmomatic and FastX Tookit, and low-quality reads (quality score Q < 30) were filtered from the data using Trim Galore! (v0.3.7) with Cutadapt (v1.9). Read alignments were performed against the Xenopus laevis genome (v.9.1) using Bowtie2 (v2.2.9), with the --very-sensitive preset option. BAM alignment files were converted into the bigWig files using bam2bigwig.
Supplementary_files_format_and_content: All processed files are in the bigWig (bw) format. Bisulfite-seq files represent methylation in CpG context and are combined for the three biological replicates for each condition. ChIP-Seq and 5hmC DIP-seq files represent alignments for each read and are provided separately for each biological replicate.
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| Submission date |
Sep 03, 2021 |
| Last update date |
Jan 07, 2022 |
| Contact name |
Ben Gregory Szaro |
| E-mail(s) |
bgs86@albany.edu
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| Phone |
518-591-8852
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| Organization name |
University at Albany, SUNY
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| Department |
Biological Sciences
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| Lab |
Life Sciences 1059
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| Street address |
1400 Washington Avenue
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| City |
Albany |
| State/province |
NY |
| ZIP/Postal code |
12222 |
| Country |
USA |
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| Platform ID |
GPL21248 |
| Series (2) |
| GSE183350 |
Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate (Xenopus laevis) Central Nervous System assayed by 5hmC MeDIP and by ChIP-seq for Histone Markers |
| GSE183357 |
Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate (Xenopus laevis) Central Nervous System |
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| Relations |
| BioSample |
SAMN21215032 |
| SRA |
SRX12007757 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5556011_ONC_H3K9me3-UO_01.bw |
1019.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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