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Sample GSM6911236

Query DataSets for GSM6911236

Status

Public on Jun 23, 2023

Title

RNA_E2C_Ctrl_rep2

Sample type

SRA

Source name

early 2-cell mouse embryos

Organism

Mus musculus

Characteristics

tissue: early 2-cell mouse embryos
antibody: none
genotype: WT

Treatment protocol

Oocytes or enbryos were isolated from 7-8-week-old female mice after intraperitoneal injection PMSG or HCG. For FGO, PMSG48h;For MII oocytes, HCG 13h;For embryos, 30h, 36h and 48h.The zona pellucida of oocytes and embryos were removed using Tyrode’s solution before they were collected.

Growth protocol

TDP-43 mice were maintained in a hybrid background of C57BL/6J and ICR under guidelines of the Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences.Oocytes and embryos were isolated in M2 medium, early embryos were culture in KOSM meduim.

Extracted molecule

total RNA

Extraction protocol

The oocytes and embryos were collected as Smart-seq2 or Stacc-seq protocols in the paper, and the extraction was amplificated or used for the library directly as in the paper.
For RNA-seq, RNA libraries for RNA-seq were prepared using a kit of Vazyme (TD502) and performed as the protocol of manufacture. For stacc-seq, Stacc-seq libraries performed according to the previous report.1 μl fully dissolved 5% digitonin was added to Buffer1 (10 mM Tris-HCl pH = 7.4, 150 mM NaCl, 0.5 mM spermidine, 1 × EDTA-free Roche complete protease inhibitor). 0.5 μl PA/G-Tn5 and 0.5 μg TDP-43 or Pol II antibody was added to prepared Buffer1 and incubated at 4℃ for 30 min. Meanwhile, oocytes and embryos were treated gently with Tyrode’s solution for removing the zona pellucida, then were put into 200ml low-binding tube contained 6ml prepared Buffer1 and incubate 4℃ for 10 min . Then 35 ml the prepared Buffer1, mixture of PA/G-Tn5, and the antibody and 12.5 ml TTBL were added for cleaving the chromatin around the binding sites and transposing with adaptors. Then the DNA were purified and PCR as the stacc-seq steps in previously reported

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

RNA_E2C_Ctrl_rep2

Data processing

Stacc-seq reads were aligned to the mm9 genome with the parameters: -t -q -N 1 -L 25 -X 2000 --no-mixed --no-discordant by Bowtie2 v2.3.5. Aligned reads were filtered with a minimum MAPQ of 20, and PCR duplicates were removed using Picard MarkDuplicates v1.119. Read coverages over mm9 genome were estimated by bamCoverage from deepTools v3.3.1 with parameters --binSize 100 --normalizeUsing RPKM.
RNA-seq reads were trimmed by Trim Galore v0.6.6 and then mapped to the mm9 genome by HISAT2 v2.2.1(Kim et al., 2019). StringTie v2.1.2(Pertea et al., 2016) was used to calculate the FPKM per gene based on mm9 refFlat annotation from the UCSC genome annotation database.
Assembly: mm9
Supplementary files format and content: Bigwig for Stacc-seq signals
Supplementary files format and content: gene expression FPKM table for RNA-seq data

Submission date

Jan 01, 2023

Last update date

Jun 23, 2023

Contact name

Fengling Chen

E-mail(s)

cfl15@tsinghua.org.cn

Organization name

Tsinghua University

Street address

30 Shuangqing Rd.