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| Status |
Public on Aug 30, 2022 |
| Title |
RNA_late2C_TSA_rep1 |
| Sample type |
SRA |
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| Source name |
late 2-cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: late 2-cell
strain: B6D2F1/J (female) x B6D2F1/J (male)
genotype: WT
treatment: treated with TSA
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| Treatment protocol |
To transiently inhibit CBP/p300, embryos were cultured in KSOM containing 10μM A-485 (Tocris) between 4 and 20hpi. For transient inhibition of HDACs, embryos were cultured in KSOM containing 50nM TSA (Sigma) between 0-20 or 8-28hpi. Embryos were washed at least six times when they were transferred between different culture conditions.
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| Growth protocol |
All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Harvard Medical School. Collection of oocytes and in vitro fertilized embryos were described previously (Chen et al., 2021; Chen & Zhang, 2019). For allelic H3K27ac CUT&RUN profiling experiments, oocytes were collected from 6- to 8-week-old B6D2F1/J female mice (Jax 100006) and sperm were collected from 8- to 10-week-old PWK/PhJ male mice (Jax 003715). For CBP/p300 and HDACs inhibition experiments, both oocytes and sperm were retrieved from 6- to 8-week-old B6D2F1/J mice. The time when capacitated sperm were added to cumulus oocyte complexes in Human Tubal Fluid (HTF, Millipore) was considered as 0 hour post in vitro fertilization (0hpi). Embryos were cultured in potassium simplex optimized medium (KSOM, Milliplore) at 37°C under 5% CO2 with air.
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| Extracted molecule |
total RNA |
| Extraction protocol |
CUT&RUN libraries were generated based on the original protocol (Meers et al, 2019) with some modifications to optimize for low-input cell numbers. For each library, 10μl BioMag Plus Concanavalin A beads (Polysciences) were washed twice in 1ml Binding Buffer (20mM HEPES-KOH pH7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2) and resuspended in 5μl Binding Buffer. Then embryos were added in 45μl Wash Buffer (20mM HEPES-NaOH pH7.5, 150mM NaCl, 0.5mM Spermidine, 1X Roche Protease Inhibitor Cocktail), mixed with the 5μl Binding Buffer containing beads and rotated for 10min at room temperature. After removing the liquid by putting the tube on the magnet stand, the beads were resuspended in 80μl antibody Incubation Buffer (1X Wash Buffer with 0.02% Digitonin and 2mM EDTA) with 1:100 diluted H3K27ac antibody (Millipore, 07-360) and rotated at 4oC overnight. Then the beads were washed twice with 100μl Wash Buffer with 0.02% Digitonin and pulled off the liquid. The beads were resuspended in 100μl Wash Buffer with 0.02% Digitonin and 500ng/ml pA-MNase, then rotated for 3h at 4oC. After washing the beads twice with 200μl Wash Buffer with 0.02% Digitonin, the beads were resuspended in 200μl ice-cold Wash Buffer with 0.02% Digitonin and 4μl 100mM CaCl2 and incubated at 0oC for 30min. The reaction was stopped by adding 23μl 10X Stop Buffer (1,700mM NaCl, 100mM EDTA, 20mM EGTA, 0.02% Digitonin, 250µg/ml RNase A, 250µg/ml Glycogen). Then the tube was incubated at 37oC for 10min to release the cut fragments into supernatant. The tube was put on a magnet stand and the supernatant was transferred to a new 1.5ml LoBind tube (Eppendorf) while the beads were discarded. 2.5μl 10% SDS and 2.5μl Proteinase K (20mg/ml) were added to the supernatant and incubated at 55oC for 1h. Then 25ng carrier RNA (Qiagen, 1068337) was added to the supernatant. The supernatant was mixed with 200μl Phenol:Chloroform:Isoamyl Alcohol (Invitrogen, 15593031) by vortexing then transferred to a phase-lock tube (QuantaBio, 2302830) and centrifuged at 16,000g for 5min at room temperature. The supernatant was transferred to a new 1.5ml LoBind tube, added 26μl 3M Sodium Acetate, 1μl Glycogen (20mg/ml) and 750μl cold 100% Ethanol, mixed by vortexing and stored at -20oC overnight. Then the tube was centrifuged at 16,000g for 20min at 4oC and the supernatant was discarded. The precipitate DNA pellet was washed with 1ml cold 80% Ethanol then centrifuged at 16,000g for 5min at 4oC and the supernatant was discarded. Finally the precipitate DNA pellet was dissolved in 25μl ultrapure H2O. The library was constructed with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S) following the manufacturer protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
For CUT&RUN sequencing data, the raw paired-end reads were trimmed with Trimmomatic (v0.39) (Bolger et al, 2014) to remove adaptors and the trimmed reads with length at least 35bp were retained. The cleaned reads were mapped to GRCm38 reference genome using bowtie2 (v2.4.2) (Langmead & Salzberg, 2012) with parameters: --local --very-sensitive-local --no-unal --no-mixed --no-discordant --dovetail -I 10 -X 700 --soft-clipped-unmapped-tlen. PCR duplicates were removed with Picard MarkDuplicates (v2.23.4). The mapped reads were further filtered to retain proper paired reads with fragment length at least 140 and mapping quality at least 30. The signal tracks were generated by bamCoverage in deeptools (v3.5.1) (Ramirez et al, 2016) with 100bp bin size and normalized with FPKM. Since the H3K27ac showed different broad or narrow patterns in different stages of early embryos, the FPKM values were not directly comparable among stages. To make the FPKM values comparable, we followed the scaling method used in the previous study of H3K4me3 (Dahl et al., 2016), which also showed broad and narrow patterns in different stages. We used the signals in ESCs as reference so the ESCs had a scale factor of 1. We ranked the gene promoters by their H3K27ac signals. Then for each stage, we chose the median signal of the top 3,000 promoters, dividing from the median signals of the top 3,000 promoters in ESCs to obtain the sale factor for that stage. The FPKM signals in each stage were multiplied by the scale factor to derive the scaled FPKM, which was used in all the downstream analysis. For allelic analysis, we used SNPsplit (v0.5.0) (Krueger & Andrews, 2016) to assign the mapped reads to maternal or paternal allele based on the SNPs information between B6D2F1and PWK/PhJ. The SNPs were downloaded from the Mouse Genomes Project (https://www.sanger.ac.uk/data/mouse-genomes-project/). The SNPs that were identical between C57BL/6J and DBA/2J while different with PWK/PhJ were used.
For the raw sequencing reads of total RNA-seq, the three leading nucleotides of read2 generated from TSO were removed. Then the adaptors were removed with Trimmomatic (v0.39) if present. The cleaned reads were mapped to GRCm38 reference genome using STAR (v2.7.8a) (Dobin et al, 2013). RSEM (v1.3.1) (Li & Dewey, 2011) was employed to quantify the genes expression levels with the transcriptome alignments generated by STAR as input. For differentially expression analysis, R package DESeq2 (v1.32.0) (Love et al, 2014) was utilized with raw counts from RSEM as input. The significantly changed genes were identified with adjusted p-value cutoff 0.05, fold change cutoff 2 and mean FPKM cutoff 1.
For analyzing ATAC-seq sequencing data, we used the ENCODE ATAC-seq pipeline (v2.1.2, https://github.com/ENCODE-DCC/atac-seq-pipeline). ATAC-seq peaks within 1kb upstream or downstream of any known TSS were treated as promoter peaks, while ATAC-seq peaks with distance greater than 2.5kb to TSS were treated as distal peaks.
Assembly: mm10
Supplementary files format and content: bigWig files contain scaled H3K27ac CUT&RUN FPKMs and ATAC-seq fold changes; txt files contain gene-level reads counts of RNA-seq; bed files contain ATAC-seq peaks
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| Submission date |
Jun 29, 2022 |
| Last update date |
Aug 31, 2022 |
| Contact name |
Yi Zhang |
| E-mail(s) |
[email protected]
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| Organization name |
Boston Children's Hospital / Harvard Medical School
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| Department |
PCMM
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| Street address |
200 Longwood Avenue WAB 149
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (1) |
| GSE207222 |
Dynamic landscapes of H3K27ac in mouse preimplantation embryos |
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| Relations |
| BioSample |
SAMN29421906 |
| SRA |
SRX15944368 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6281818_RNA_late2C_TSA_rep1.gene_counts.txt.gz |
2.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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