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| Status |
Public on Jun 13, 2022 |
| Title |
PP_A5_KO_m6A_rep2 |
| Sample type |
SRA |
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| Source name |
MEL1 hESC line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MEL1 hESC line
cell type: hPSC-derived pancreatic progenitor cells
genotype: A5_KO
rip antibody: m6A (Millipore)
batch: 2
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| Growth protocol |
Undifferentiated MEL1 human embryonic stem cells were cultured in hPSC medium: DMEM/F12 (Life Technologies) supplemented with 20% KnockOut Serum Replacement (KSR) (Life Technologies), 1× Non-Essential Amino Acids (NEAA) (Life Technologies), 1× Penicillin/Streptomycin, 0.055 mM 2-Mercaptoethanol (Sigma), and 10 ng/ml bFGF (Peprotech). hPSCs were maintained on CF1 feeder cells in humidified incubator at 37°C and 5% CO2. hPSCs were seeded into 10-cm dishes and cultured for four days before differentiation. The medium was changed every day. Then, hPSCs were washed by DPBS (Life Technologies) followed by dissociated into single cells using Accutase. hPSCs were seeded into 12-well plates at a density of 5×105 per well and the differentiation was initiated 24 hours after seeding. hPSCs were quickly washed by DPBS (Life Technologies) and exposed to differentiation media. Here is the detailed media: Day 1: RPMI (Life Technologies), 1× Penicillin/Streptomycin, 100 ng ml-1 activin A, and 3 μM CHIR99021 (TargetMol). Day 2: RPMI, 0.2% FBS, 1× Penicillin/Streptomycin, and 100 ng ml-1 activin A. Day 3: RPMI, 2% FBS, 1× Penicillin/Streptomycin, and 100 ng ml-1 activin A. Day 4-6: RPMI, 0.5X B27, 0.5X N2, 0.05% BSA (Yeasen), 1× Penicillin/Streptomycin, and 50 ng ml-1 KGF (Peprotech). Day 7-8: DMEM, 1X B27 (Gibco), 0.05% BSA, 1× Penicillin/Streptomycin, 0.25 mM vitamin C, 50 ng ml-1 KGF, 0.1 μM LDN-193189 (Tocris), 0.1 μM GDC-0449 (Selleck), and 2 μM retinoic acid (Sigma). Day 9-14: DMEM, 1X B27, 0.05% BSA, 1× Penicillin/Streptomycin, 0.25 mM vitamin C, 0.1 μM LDN-193189, and 50 ng ml-1 EGF (Peprotech). Then, cells were suspended to form aggregates, and differentiated into hILO stage using medium modified from our previous protocol. Day 15-22 (R6 medium): DMEM, 1:50 B27, 0.05% BSA, 1× Penicillin/Streptomycin, 10 μM zinc sulfate (Sigma), 10 μg ml-1 heparin (Sigma), 10 μM 616452 (TargetMol), 1 μM T3 (Sigma), 0.1 μM LDN-193189, 0.1 μM compound E (MedChem Express), and 0.25 mM vitamin C. Day 23-30 (R7 medium): DMEM, 1:50 B27, 0.05% BSA, 1× Penicillin/Streptomycin, 10 μM zinc sulfate, 10 μg ml-1 heparin, 10 μM 616452, 1 μM T3, 1 mM N-acetyl cysteine (Sigma), 1 μM trolox (Sigma), and 0.25 mM vitamin C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted with TRIzol™ Reagent (Invitrogen, cat. 15596018), and mRNAs were further enriched by GenElute mRNA Miniprep Kit (Sigma, cat. MRN10–1KT).
For samples from hPSC, DE, PP and hILO stages, mRNAs were fragmented into about 100 nt and immunoprecipitated (IP) with m6A antibody (SYSY, cat. 202003), both input and IP products were subjected to library construction (Illumina). For samples from WT and A5-KO PP cells, total RNAs were extracted, fragmented and subjected to immunoprecipitation directly due to limited materials. rRNAs in input fragments were first depleted by rRNA Depletion Kit (NEB, cat. E7400L), then both input and IP fragments were subjected to library preparation using SMARTer® Stranded Total RNA-Seq Kit v2 (Takara).
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Description |
KO.m6A-peaks.txt
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| Data processing |
Illumina Casava1.8 software used for basecalling.
Sequenced reads were first trimmed by fastp (Chen et al., 2018) to remove adapter sequence and reads with low sequencing quality.
Clean data were aligned to human reference genome version 38 (GRCh38) using hisat2 (Kim et al., 2015) with parameters: --rna-strandness=FR -k 1 --no-unal.
The m6A peaks were called using R package exomePeak2 (Wei, 2020) (IP/input > 1.5, p value < 10-5), the longest isoform was retained if a gene has more than one isoform.
The differential m6A peaks were calculated using R package exomePeak2 with parameters: |log2 FC| > 1, adjust p value < 0.05.
Motif enrichment was done using HOMER (Heinz et al., 2010) selecting a motif length of 5,6 and 7 nucleotides.
Genome_build: hg38
Supplementary_files_format_and_content: m6A peak text files
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| Submission date |
Dec 29, 2020 |
| Last update date |
Jun 13, 2022 |
| Contact name |
Saiyong Zhu |
| E-mail(s) |
0015071@zju.edu.cn
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| Organization name |
Zhejiang University
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| Department |
Life Sciences Institute
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| Street address |
866 Yuhangtang Rd.
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| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310058 |
| Country |
China |
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| Platform ID |
GPL20795 |
| Series (2) |
| GSE163963 |
N6-methyladenosine mRNA Modification Is Essential for Human Pancreatic Lineage Specification [m6A-Seq] |
| GSE163964 |
N6-methyladenosine mRNA Modification Is Essential for Human Pancreatic Lineage Specification |
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| Relations |
| BioSample |
SAMN17175115 |
| SRA |
SRX9748382 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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