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| Status |
Public on Jan 27, 2022 |
| Title |
NOME-seq of Nng2 overexpressing cells rep2 |
| Sample type |
SRA |
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| Source name |
Electroporated cells E15.5 somatosensory cortex
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: E15.5
celltype: GFP+
technique: NOME-seq
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The somatosensory cortex of in utero electroporated E14.5 mouse embryos was isolated and the electroporated area dissected. Single cell suspension was prepared using the papain-based neural dissociation kit (Miltenyi Biotec, Cat. N: 130-092-628) according to the manufacturer protocol with reduced dissociation times (5 min. instead of 15 min. at 37˚C). Cell number as well as the viability was assessed using the Countess™ II Automated Cell Counter (Invitrogen). Dissociated cells were fixed for 10 min. in 1% Formaldehyde (ThermoFisher, Cat. N: 28908), quenched by addition of Glycine (Invitrogen, Cat. N: 15527013) to a final concentration of 0.2M, washed in PBS with 0.1% BSA, passed through a 40µM cell strainer (ThermoFisher, Cat. N: 15342931) and immediately FAC-sorted at a BD FACSAria Fusion (BD Bioscience) with four lasers (405, 488, 561, 640) using a 100µm nozzle. Sorted cells were lysed with 0.2% Igepal-CA630 (Sigma-Aldrich, Cat. N: I3021) and incubated for 3h at 37˚C in a reaction mix containing 4.5U/µl M.CviPI (New England Biolabs, Cat. N.: M0227S) and 0.6mM SAM (New England Biolabs, Cat. N.: B9003). During the incubation the reaction was substitution with 4U M.CviPI and 0.5µl of 32mM SAM every hour. Thereafter nuclei were lysed and reverse-crosslinked overnight at 68˚C, purified by ethanol precipitation and sheared to ~550 bp fragments using a Covaris S220 sonicator. Methylated and unmethylated spike in controls were added to the samples (~ 0.05%) prior bisulfite conversion using the the EZ DNA Methylation-Gold kit (Zymo Research, Cat. N: D5005).
Final libraries were constructed using the Accel-NGS® Methyl-Seq DNA Library kit (Swift Bioscience, Cat. N: 30024) according to the manufacturer’s instructions until the final library amplification step. To increase the complexity of the samples, library amplification was performed in 5 separate reaction using the EpiMark Hot Start Taq (New England Biolabs, Cat. N: M0490S) with Methyl-Seq Indexing Primers (Swift Bioscience, Cat. N: 36024) using the following PCR program: 95˚C 30s; {95˚C 15s, 61˚C 30s, 68 ˚C 60s} x11; 68˚C 5min; Hold at 10˚C. Different PCR reactions of a sample were pooled and purified using 0.8x AMPure XP beads (Agencourt, Cat. N: A63881).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
processed data: Bigwig files with CpG or GpC methylation levels of all cytosines covered by at last 5 reads (5x coverage).
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| Data processing |
library strategy: Nucleosome Occupancy and Methylome Sequencing (NOMe-seq)
Nome-seq reads were first trimmed using trim galore in a paired-end mode with –clip_R1=1 and –clip_R2=15 to account for the A-tail deposited by adaptase. Reads were then mapped using bismark in paired end mode, deduplicated and methylation calls were extracted (--ignore 6 and --CX). Coverage files were then produced using coverage2cytosine in a --nome-seq mode. Only calls with at least 5x coverage per replicate retained.
Genome_build: mm10
Supplementary_files_format_and_content: Compressed text file, containing pair-wise contact infomration in the medium juicer format (https://github.com/aidenlab/juicer/wiki/Pre) but omitting the read name: <str1> <chr1> <pos1> <frag1> <str2> <chr2> <pos2> <frag2> <mapq1> <mapq2>.
Supplementary_files_format_and_content: Bigwig files with CpG or GpC methylation levels of all cytosines covered by at last 5 reads (5x coverage).
Supplementary_files_format_and_content: Tab seperated table containing read counts per gene.
Supplementary_files_format_and_content: Compressed files contain sequences of selecred CREs (design.fa.gz), their annotation (labels.tsv.gz) and a pickle library with filtered CRE - Barcode associations (CRE_BC_association.pickle.gz).
Supplementary_files_format_and_content: Compressed tarball archive containing DNA/RNA read counts and annotations for both replicates.
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| Submission date |
Sep 16, 2021 |
| Last update date |
Jan 27, 2022 |
| Contact name |
Boyan Bonev |
| E-mail(s) |
[email protected]
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| Organization name |
Helmholtz Zentrum München
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| Lab |
Bonev Lab
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| Street address |
Ingolstädter Landstr. 1
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| City |
Neuherberg |
| State/province |
Deutschland |
| ZIP/Postal code |
85764 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE155677 |
Multimodal profiling of the transcriptional regulatory landscape of the developing mouse cortex identifies Neurog2 as a key epigenome remodeler |
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| Relations |
| BioSample |
SAMN21463014 |
| SRA |
SRX12214121 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5583716_NGN2_NOME_IUE24h_rep2_CpG_cov5x.bw |
95.5 Mb |
(ftp)(http) |
BW |
| GSM5583716_NGN2_NOME_IUE24h_rep2_GpC_cov5x.bw |
718.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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