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| Status |
Public on Sep 05, 2023 |
| Title |
EPSC_100K_Input2 |
| Sample type |
SRA |
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| Source name |
E14
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14
cell type: Expanded potential stem cells
antibody: none
replicate: 2
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| Extracted molecule |
total RNA |
| Extraction protocol |
For bulk m6A-CT library preparation, E14, EPSC, MEF and HEK293T nuclei were isolated with the following conditions: 1 min on ice in 0.020 % Triton X-100 for E14, 0.015 % Triton X-100 for EPSC; 5 min on ice in 0.2 % NP-40 for MEF, 0.02 % NP-40 for HEK293T. Aliquots of nuclei (5,000–100,000 nuclei) were bound to pre-activated Concanavalin A (Con-A) Conjugated Paramagnetic Beads (EpiCypher, #21-1411) and used in assay. All wash steps were performed on a magnetic separation rack. Undirected cDNA/RNA hybrid tagmentation for INPUT RNA was carried out as described previously. Briefly, nuclei were resuspended in 50 μL Reverse Transcription & Tagmentation Buffer, and incubated for 1 hour at 37 °C. 2.5 μL pAG-Tn5 was then added and vortexed briefly, and incubated for 1 hour at 37 °C. Antibody directed tagmentation for m6A-CT was carried out as same as sc-m6A-CT using 1 μg anti-m6A antibody (Synaptic Systems, #202003). When tagmentation was done, 54.5 μL of 0.5 M EDTA, 1 μL of 20 mg/mL Proteinase K and 5.5 μL of 10% SDS were sequentially added to the sample and incubated at 58 °C for 1 hour. An equal amount of Phenol-chloroform-isoamyl alcohol 25:24:1 (PCI), pH 6.7 was added to each sample, and PCI extraction followed by ethanol precipitation were performed. To perform gap-filling, the purified RNA/DNA samples were mixed with 0.32 U/μL Bst 3.0 DNA Polymerase (New England Biolabs) and Q5 High-Fidelilty 2× PCR Master mix (New England Biolabs), and incubated for 5 min at 58 °C, 5 min at 72 °C, and then at 5 min at 80 °C to inactivate Bst 3.0. A qPCR cycle check can be performed at this stage using 1:100 templates. A successful experiment can be identified by comparing cycle threshold values to IgG-CT. A universal i5 and a uniquely barcoded i7 primer, and Q5 HiFi 2× PCR Master mix were used to amplify libraries with the following cycling conditions: 45 sec at 98 °C, followed by 14-21 cycles of 15 sec at 98 °C and 10 sec at 60 °C, with a final extension at 1 min at 72 °C. Post-PCR clean-up was performed by 0.5× right-side followed by 1.2× left-side SPRI beads (Beckman Counter) selection.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Adapter sequences of paired-end raw reads were trimmed using Trim Galore with parameters -q 30.
Trimmed reads were aligned to the hg19 & mm9 genome assembly using STAR aligner version 2.7.2 with the following parameters: --outFilterMatchNminOverLread 0 --outFilterScoreMinOverLread 0 --outFilterMatchNmin 30 --outFilterMismatchNmax 3 --outFilterMultimapNmax 1.
Peaks were called using Macs2 with parameters -f BAM -B -q 0.01 --nomodel --extsize 100 --keep-dup all and called peaks with log-q-value > 10 were used in downstream analyses.
Assembly: hg19 and mm9
Supplementary files format and content: Bed files were narrowPeak files generated by Macs2.
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| Submission date |
Nov 04, 2022 |
| Last update date |
Sep 05, 2023 |
| Contact name |
Kiyofumi Hamashima |
| Organization name |
Institute of Molecular and Cell Biology (IMCB)
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| Lab |
Epigenetics and Cell Fates Laboratory
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| Street address |
61 Biopolis Drive
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| City |
Singapore |
| ZIP/Postal code |
138673 |
| Country |
Singapore |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE217254 |
Single-nucleus multiomic mapping of m6A methylome and transcriptome in native populations of cells with sn-m6A-CT [Bulk CUT&Tag] |
| GSE217256 |
Single-nucleus multiomic mapping of m6A methylome and transcriptome in native populations of cells with sn-m6A-CT |
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| Relations |
| BioSample |
SAMN31605121 |
| SRA |
SRX18169191 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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