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| Status |
Public on Dec 04, 2023 |
| Title |
4C_WT,rep1 |
| Sample type |
SRA |
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| Source name |
Hippocampus
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| Organism |
Mus musculus |
| Characteristics |
tissue: Hippocampus
Sex: Male
genotype: WT
treatment: Tamoxifen
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For RNA, total RNA was extracted from hippocampal tissue with TRI reagent (Sigma-Aldrich) as recommended by the manufacturer. Subsequently, genomic DNA was eliminated by a treatment with DNAse I (Qiagen) for 30 min at 25ºC. Followed by precipitation with phenol-chloroform-isoamyl alcohol (Sigma-Aldrich).
ChIPs were performed as previously described in detail (Galvão-Ferreira et al., 2017) with minor modifications. Briefly, 3 μg of antibody were incubated with Dynabeads coated to Protein G (Invitrogen) in RIPA-150 buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM, EDTA, 0.1% SDS, 1% Triton X-100, and 0.1% sodium deoxycholate; pH 8) for 6-18 h at 4°C in agitation. Nuclei from hippocampal cells were extracted in a dounce homogenizer as explained for the FANS method. Samples were fixed with 1% formaldehyde (Sigma-Aldrich) for 10 min at room temperature followed by 0.1 M glycine for 5 min to stop the fixation. Nuclei were centrifuged at 1,5 G, and the pellet was resuspended in 100 μL of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris; pH 8) prior to its sonication in a Bioruptor Pico (Diagenode) for 12 cycles of 15 s on/30 s off. After 6 min of centrifugation at 13000 rpm, the supernatant containing isolated chromatin was collected. Samples were diluted in 900 μL of ChIP Dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl; pH 8) and incubated overnight at 4°C with the antibody-Dynabeads mix. Beads were thoroughly rinsed at 4ºC as follows: two washes in RIPA-150 buffer, three washes in RIPA-500 buffer (50mM Tris-HCl, 500 mM NaCl, 1 mM, EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate; pH 8), two washes in RIPA LiCl buffer (50 mM Tris-HCl, 1 mM EDTA, 1% NP-40, 0.7%, sodium deosycholate, 500 mM LiCl2), and two final washes in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA; pH 8.0), five minutes each. Samples were resuspended in 200 μl Elution buffer (10 mM Tris-HCl, 5 mM EDTA, 300 mM NaCl, 0.5% SDS; pH 8) and treated with 1 μl of 10 mg/mL RNase A (Fermentas) overnight at 65°C in agitation for crosslink reversion. Samples were treated with 3 μl of 20 mg/mL Proteinase K (Thermo Scientific) for 2 hours at 55°C in agitation. Last, DNA precipitation was performed by phenol-chloroform-isoamyl alcohol (Sigma-Aldrich). For Kdm1a ChIP, some modification were applied as in (Lipinski et al., 2020). In brief, samples required a stronger fixation treatment in 1% PFA for 30 min at 37ºC. To fragment these highly fixed samples, 43 cycles of sonication (30 s On, 30 s Off) were applied in a Bioruptor Pico (Diagenode). SDS lysis buffer was modified to RIPA buffer (0.1% SDS, 1% IGEPAL, and 0.5% sodium deoxycholate).
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) using a CTCF-specific antibody (Abclonal Cat # ab70303) was performed as described in the in situ ChIA-PET protocol (Wang et al., 2021) on approximately 10 million nuclei of hippocampal excitatory neurons sorted by FANS as described above.
4C-seq, was performed as previously described (Krijger et al., 2020) with minimal changes. Approximately 5 million hippocampal nuclei were fixed at 1 % with PFA. The restriction enzyme cutting order was Dpn II as the primary enzyme and Csp6I as the secondary enzyme. Ligated chromatin was phenol-chloroform extracted and ethanol precipitated. Reactions were then further purified with Ampure XP beads with a 0.8x ratio of bead solution to library following the manufactures instructions. Samples were then quantified with Qubit and 4C-seq libraries were sequenced using an Illumina high-throughput sequencing equip.
For RNA three mice were used per genotype (Kdm1a-ifKO and their control littermates) and Poly-A libraries were generated. For RNA and ChIPseq, libraries were generated for each independent sample and single-end sequenced in a HiSeq 2500 apparatus from Illumina. For ChIAPet, librearies were generated and paired end and 150 pb were sequenced and emrged from Miseq, Nextseq 500/550 and Hiseq 3000/4000 aparatus.
PolyA RNAseq, ChIP-seq, 4C-seq and ChIAPet
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
For RNA, adapters were trimmed using cutadapt v1.18 selecting reads longer than 25 bp. HISAT2 (Kim et al., 2015) (v2.1.0) was used for reads alignment to the mouse genome (GRCm38.89, mm10) and reads with mapq > 30 and that mapped to nuclear chromosomes were quantified using HTSeq v0.11.1 (Anders et al., 2015). Data processing was performed with custom R scripts (v3.5.1, 2018), Samtools v1.9 (Li et al., 2009), Bedtools v2.27.1 (Quinlan and Hall, 2010), and DeepTools v3.5.0 (Ramírez et al., 2016). Whole genome alignments were normalized to 10× RPM (read per 10 million sequenced reads).
For ChIPs, Adapters were trimmed using TrimGalore v 0.6.4_dev (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), and the reads obtained were mapped to the mouse genome (mm10) with Bowtie2 (v2.3.4.3) (Langmead and Salzberg, 2012). Only those reads with a mapq > 30 were selected. Data was processed using Samtools v1.10 (Li et al., 2009), Deeptools v 3.5.0 (Ramírez et al., 2016), and Bedtools v2.25.0 (Quinlan and Hall, 2010). For BigWigs, ChIP-seq reads were normalized by reads per genomic content with Deeptools (Ramírez et al., 2016).
ChIA-PET, was processed by the ChIA-PIPE pipeline (Lee et al., 2020) using mm10 as a reference genome and default parameters. The resulting *hic file for 2D contact maps and *bigwig file for protein binding coverage were used for visualizing on Juicebox (Durand et al., 2016) Within ChIA-PIPE, peaks were called via MACS2 without input control and only those with the maximum coverage greater than 50 (which is twice the median of all peaks) were kept by pybedgraph (Zhang et al., 2020), resulting in 34,885 confident peaks. Using these peaks, the loops reported in *bedpe format with 7th column denoting PET counts are further filtered to have PET count greater than or equal to 3 and to have both left and right anchors to overlap with at least one peak.
For 4C-seq analysis all samples were trimmed to 75 bp. The wig and peaks were calculated using pipe4C and peakC as mentioned in (Krijger et al., 2020).
Assembly: mm10
Supplementary files format and content: For RNA tab-delimited text file contains raw counts data for each sample, BigWig files
Supplementary files format and content: BigWig files for ChIP-seq data
Supplementary files format and content: BigWig, Bedgraph and hic files for ChIAPet data
Library strategy: 4C-seq
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| Submission date |
Jun 29, 2023 |
| Last update date |
Dec 04, 2023 |
| Contact name |
Sergio Niñerola |
| E-mail(s) |
sninerola@umh.es
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| Phone |
+34646912958
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| Organization name |
CSIC-Instituto de Neurociencias de Alicante
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| Department |
Molecular Neurobiology and Neuropathology
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| Lab |
Transcriptional and epigenetic mechanisms of neuronal plasticity
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| Street address |
Avenida Santiago Ramon y Cajal, s/n
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| City |
San Juan de Alicante |
| State/province |
Alicante |
| ZIP/Postal code |
03550 |
| Country |
Spain |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE236182 |
Kdm1a safeguards the topological boundaries of PRC2-repressed genes and prevents aging-related euchromatinization in neurons |
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| Relations |
| BioSample |
SAMN36084891 |
| SRA |
SRX20833439 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7519437_1WT_spint_WIN21.wig.gz |
24.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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