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Sample GSM2182643

Query DataSets for GSM2182643

Status

Public on Oct 27, 2016

Title

Tet-MII-TKO2

Sample type

SRA

Source name

Tet-MII-TKO

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