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Sample GSM6710118 Query DataSets for GSM6710118
Status Public on Sep 05, 2023
Title E14_25K_m6A-CT2
Sample type SRA
 
Source name E14
Organism Mus musculus
Characteristics cell line: E14
cell type: Mouse embryonic stem cells
antibody: m6A (Synaptic systems, #202003)
replicate: 2
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.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
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.
 
Submission date Nov 04, 2022
Last update date Sep 05, 2023
Contact name Kiyofumi Hamashima
Organization name Institute of Molecular and Cell Biology (IMCB)
Lab Epigenetics and Cell Fates Laboratory
Street address 61 Biopolis Drive
City Singapore
ZIP/Postal code 138673
Country Singapore
 
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
Relations
BioSample SAMN31605131
SRA SRX18169181

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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