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Sample GSM2028283 Query DataSets for GSM2028283
Status Public on Oct 27, 2016
Title Tet_TKO1_E6.5_Epiblast_PBAT
Sample type SRA
Source name Epiblast of E6.5
Organism Mus musculus
Characteristics tissue: Epiblast
age: Embryonic day 6.5
genotype: Tet1-/- Tet2-/- Tet3-/-
strain: C57BL/6J-129Sv
Treatment protocol A pool of 23 WT and 26 Tet-null fresh epiblasts were disrupted and homogenized in Buffer RLT Plus respectively, then snap-frozen in liquid nitrogen and stored at -80 ºC.
Growth protocol For embryo preparation, mice were maintained on a 12/12 h light/dark cycle. After mating, the morning of appearance of a vaginal plug was designated as embryonic day 0.5 (E0.5). On day 7 of pregnancy, corresponding to E6.5, embryos were recovered carefully from uteri and isolated from decidua using fine forceps and a needle under a dissecting microscope. After incubation in PBS with 0.25% pancreatin (Sigma, P7545) and 0.05% trypsin (Gibco, 27250-018) for 10 minutes at 4 °C, embryos were transferred to Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) supplemented with 10% FBS and then the visceral endoderm layer was detached from embryos by being gently aspirated through a mouth pipette several times. The epiblast was separated from the extraembryonic ectoderm using a fine tungsten needle. The visceral endoderm layer or the extraembryonic ectoderm was used for genotyping.
Extracted molecule genomic DNA
Extraction protocol DNA was extracted from isolated epiblasts by using the Qiagen AllPrep DNA/RNA Mini Kit.
About 5 ng genomic DNA were used to construct the PBAT library using the protocol slightly modified from previously published. Briefly, the isolated genomic DNAs, together with 1% unmethylated lambda DNA (Thermo Scientific), were converted and purified with MethylCode Bisulfite Conversion kit (Invitrogen) following the manufacturer’s instructions. Then random nonamer primers with a 5’ biotin tag and a truncated Illumina P5 adaptor (5’-biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3’) and 50 units of Klenow polymerase (3’ to 5’ exo-, NEB) were used to amplify the bisulfite-converted templates linearly. The excess primers remained in last reaction were removed using 40U Exonuclease I (NEB) before amplified products were purified with 0.8 x Agencourt Ampure XP beads (Beckman Coulter). The newly synthesized DNA strands with biotin were captured with Dynabeads M280 Streptavidin (Invitrogen), and the original bisulfite-treated DNA templates were washed away by 0.1 N NaOH. The second strands were synthesized using 50 units Klenow polymerase with random nonamer primers containing a truncated P7 Illumina adaptor (5’-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3’). The beads with double strand DNA products were further collected and washed several times, and the libraries were generated with 5-6 cycles of PCR amplifications using 1 unit KAPA HiFi HS DNA Polymerase (KAPA Biosystems), together with 0.4 μM Illumina Forward PE1.0 primer (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and 0.4 μM pre-indexed Illumina Reverse primer (5’- CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’; the underlined hexamer indicates the index sequences). Amplified libraries were purified with 0.8 x Agencourt Ampure XP beads twice and were assessed with the Fragment Analyzer (Advanced analytical) and quantified with a standard curve-based qPCR assay (Roche). The final quality-ensured libraries were pooled and sequenced on the Illumina X Ten sequencer for 150 bp paired-end sequencing.
whole-genome bisulfite sequencing by post-bisulfite adaptor tagging (PBAT)
Library strategy OTHER
Library source genomic
Library selection other
Instrument model HiSeq X Ten
Data processing library strategy: whole-genome bisulfite sequencing by post-bisulfite adaptor tagging (PBAT)
For data from Illumina, Illumina CASAVA version 1.8 were used to the basecalling.
Adaptor contamination and low-quality reads were discarded from the raw data.
For RNA-Seq Illumina paired-end reads, TopHat (version 2.0.10) were used for sequence alignment, and nCount values were generated by HTSeq(version 0.6.1) and then normalized by DESeq2(version 1.6.1).
For PBAT DNA illumina paired-end reads,Bismark(version 0.7.6) were use for sequence alignment, and SingleC data were generated by SAMtools(version 0.1.19-44428cd)
Bisulfite conversion rate was estimated by the lambda genome, which was built as the extra chromosome.
Genome_build: mm9, J02459.1
Supplementary_files_format_and_content: SingleC data were generated by SAMtools(version 0.1.19-44428cd)
Submission date Jan 06, 2016
Last update date May 15, 2019
Contact name Rui WANG
Phone 15801166445
Organization name Peking University
Department Biodynamics Optical Imaging Center (BIOPIC)
Lab Fuchou Tang
Street address No.5 Yiheyuan Road, Haidian District
City Beijing
ZIP/Postal code 100871
Country China
Platform ID GPL21273
Series (1)
GSE76261 DNA demethylation by Tet dioxygenases controls gastrula patterning by regulating Lefty-Nodal signaling
BioSample SAMN04386718
SRA SRX1519759

Supplementary file Size Download File type/resource
GSM2028283_Tet_TKO1_E6.5_Epiblast.SingleCmet.txt.gz 2.9 Gb (ftp)(http) TXT
GSM2028283_Tet_TKO1_E6.5_Epiblast.SingleCmet_lambda.txt.gz 418.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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