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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 30, 2019 |
| Title |
RNAseq_2i_Day3_Rep1 |
| Sample type |
SRA |
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| Source name |
Mouse Embryonic Stem Cells
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| Organism |
Mus musculus |
| Characteristics |
background strain: E14
cell type: Mouse Embryonic Stem Cells
treatment: 2i
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| Growth protocol |
E14Tg2a ESCs were cultured in standard culture medium on gelatin coated-dishes without feeder cells. For serum culture, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum, L-glutamine (2 nM), Na-Pyruvate (1 mM), non-essential amino acids (0.1 mM each), Penicillin Streptomycin, 2-mercaptoethanol (55 µM) and LIF (1000 U/ml, Milipore) was used. For serum free culture, cells were transferred from serum-medium to NDiff 227 (StemCells, Inc.) supplemented with MEK inhibitor PD0325901 (1 μM) and GSK3 inhibitor CHIR99021 (3 μM) and LIF (1000 U/ml, Milipore) or maintained for long term (>4 weeks) in 2i medium (2i_LT).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was performed using 8 time points: Day1, Day3 and Day7 of serum-2i and 2i-serum transitions and both steady-states (serum and 2i) samples. RNA isolation was performed using Qiagen RNeasy protocol and 4 microgram RNA was employed for generating the RNA-seq libraries. Total RNA was subjected to rRNA depletion using the Ribo-ZeroTM Gold kit (Epicentre) according to manufacture instruction. rRNA-depleted samples were employed in first and second strand cDNA synthesis and by using dUTP for strand-specificity. Sequencing libraries were then prepared using Kapa-Hifi (KAPA Biosystems) according to manufactory’s instruction and the final libraries were sequenced on HiSeq 2000 Illumina sequencer.
Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 minutes at 20°C and then for 30 minutes at 65°C.Subsequently adapters were ligated by adding 30µl ligation buffer, 10 Kapa l DNA ligase, 5µl diluted adaptor in a total volume of 110µl and incubated for 15 minutes at 15°C.Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20µl elution buffer. Libraries were amplified by adding 25µl 2x KAPA HiFi Hotstart ReadyMix and 5µl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and 300bp fragments selected using E-gel. Correct size selection was confirmed by BioAnalyzer analysis. Sequencing was performed using Illumina HiSeq 2000 machines and generated 43bp single end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
FASTQ files against mouse rRNA, tRNA, snRNA, snoRNA, mtRNA sequences using Bowtie (Ref) to deplete these small RNA contamination in sequenced libraries. We then mapped the cleaned files against mouse genome (mm9) using GSNAP (Ref) and by applying the following parameters: -B 5 -t 15 -N 1 -E 100 -w 100000 -n 10 -s mm9refGene_splice.
Genome_build: mm9
Supplementary_files_format_and_content: BigWig tracks, data are not normalized for sequencing depth.
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| Submission date |
Dec 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hendrik G Stunnenberg |
| E-mail(s) |
h.stunnenberg@ncmls.ru.nl
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| Organization name |
Radboud University
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| Street address |
Geert Grooteplein 30
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| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE92403 |
Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. (RNA-seq) |
| GSE92412 |
Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. |
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| Relations |
| BioSample |
SAMN06141912 |
| SRA |
SRX2423156 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2429114_5696.bw |
55.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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