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Sample GSM2182645 Query DataSets for GSM2182645
Status Public on Oct 27, 2016
Title Tet-MII-WT2
Sample type SRA
 
Source name Tet-MII-WT
Organism Mus musculus
Characteristics strain background: C57BL/6J-129Sv
genotype/variation: Tet1+/+ Tet2+/+ Tet3+/+
age: MII oocyte
tissue: oocyte
Treatment protocol A pool of 200 (x 2) WT and 200 (x 2) Tet-null fresh oocytes were disrupted and homogenized in Buffer RLT Plus respectively, then snap-frozen in liquid nitrogen and stored at -80 ºC.
A pool of 46 WT and 46 Tet-null fresh blastocysts were disrupted and homogenized in Buffer RLT Plus respectively, then snap-frozen in liquid nitrogen and stored at -80 ºC.
Growth protocol For collection of mature oocytes, oviducts were removed from the female mice 13–15h after human chorionic gonadotropin injection. Cumulus–oocyte complexes were released into HCZB containing 0.1% bovine testicular hyaluronidase (300 USP units per mg; ICN Biomedicals Inc.).
Cavitated E3.5 blastocysts were flushed from the uteri of naturally mated mice into M2 or DMEM followed by sequential washing in KSOM.
Extracted molecule polyA RNA
Extraction protocol Total RNA was extracted using Rneasy Micro Kit (QIAGEN, Cat No. 74004), and treated with Rnase-Free Dnase Set (QIAGEN, Cat No. 79254).
Total RNA was purified from isolated blastocysts by using the Qiagen AllPrep DNA/RNA Mini Kit, treated with DNase.
Then libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Cat No. E7530L) following the manual. Then libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Cat No. E7530L) following the manual.
About 1 ng of extracted RNA was first amplified 18 cycles according to the protocol for single-cell RNA-seq published previously. Briefly, 1 ng of extracted RNA was reverse translated into cDNA, then the free primers were removed by the ExoSAP-IT. TdT was used to add PolyA to the 3’ end of cDNA, the second strand were synthesized, and the double-stranded DNA product were amplified 18 cycles to obtain enough product for DNA library construction. The amplified DNA product was purified by DNA Clean & Concentrator™-5 (Zymo Research), and further purified by Zymoclean™ Gel DNA Recovery Kit (Zymo Research) for the 0.5-8 kb size DNA products. The gel purified products were sheared by Covaris S2 for the generation of fragments about 250 bp in length, and purified with DNA Clean & Concentrator™-5 (Zymo Research). Then NEBNext® UltraTM DNA Library Prep Kit for Illumina® (NEB, Catalog #E7370L) was used to construct a cDNA library, with reagent amount reduced to half of the reactions. The cDNA libraries were sequenced on Illumina HiSeq 2500 instrument with 100-bp reads and paired-end parameter.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model HiSeq X Ten
 
Description processed data file: MII_ncount.txt.gz
Data processing 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)
Genome_build: Reference sequence and transcript annotation files were from UCSC genome assembly mm9. Bisulfite conversion rate was estimated by the lambda genome, which was built as the extra chromosome.
Supplementary_files_format_and_content: nCount data were generated by DESeq2 and SingleC data were generated by SAMtools (version 0.1.19-44428cd)
 
Submission date May 31, 2016
Last update date May 15, 2019
Contact name Rui WANG
E-mail(s) fish_cat_wr@sina.cn
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
Relations
BioSample SAMN05189973
SRA SRX1809899

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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