 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 19, 2020 |
| Title |
siDnmt1-RNAseq |
| Sample type |
SRA |
| |
|
| Source name |
embryos
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
tissue: embryos
developmental stage: 4/8-cell
sirna: siDnmt1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Due to the limited starting amount of materials, we adopted pico-profiling method as described in previous reports to enrich the transcriptomic materials. First, mRNAs were isolated from a pool of si-Dnmt1 or si-Control injected 4/8-cell embryos using Dynabeads mRNA DIRECT Kit (Invitrogen). Using the extracted mRNAs as templates, pico-profiling specific adapter-tagged ds-cDNA fragments were generated by reacting mRNA and pico-profiling primers with SuperScript III (Invitrogen) followed by T4 DNA polymerase (NEB). Then, the adapter-tagged ds-cDNAs were amplified by 20 cycles of PCR using the anchor primers for the tagged adapters. The amplicons were digested by MlyI (NEB) to remove the pico-profiling adapters, and then the products were purified using AMPure XP beads (Beckman).
RNA-seq libraries were generated using the pico-profiling products according to the protocol suggested by NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) and home-brew method previously reported. If not indicated otherwise, all materials were purchased from NEB. Two hundred-ng pico-profiling products were treated with T4 DNA polymerase, T4 polynucleotide kinase, and Klenow enzymes to repair the ends of amplified DNA fragments. The reaction products were purified using AMPure XP Beads (Beckman Coulter) according to manufacturer’s instructions. To adenylate 3’-ends of the end-repaired products, DNAs were reacted with Klenow Fragment (3'→5' exo) and 10 mM dATP for 2 hours at 30°C. After purification with AMPure XP Beads, NEBNext adapters with unique bar-codes were ligated overnight at 16°C in the presence of diluted T4 DNA ligase. The next day, ligation mixtures were purified twice with AMPure XP Beads and were enriched by PCR. Paired-end sequencing was performed using NextSeq 550 (Illumina) to generate raw sequencing data.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
processed data file: DESeq2_counts_normalized.csv
|
| Data processing |
Raw RNA-seq reads were trimmed using Trim_galore.
Trimmed reads were mapped on mm9 using HISAT2.
Gene expression levels were calculated by HTSeq.
Differential expression analysis was performed using DESeq2.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: DESeq2_counts_normalized.csv: DESeq2 normalized expression of si-Dnmt1 or si-Scrb injected embryos.
|
| |
|
| Submission date |
Feb 28, 2018 |
| Last update date |
Jul 19, 2020 |
| Contact name |
Byungkuk Min |
| E-mail(s) |
mbk0asis@gmail.com
|
| Organization name |
KRIBB
|
| Department |
Development and Differentiation Research Center
|
| Street address |
125 Gwahak-ro, Yuseong-gu
|
| City |
Daejeon |
| ZIP/Postal code |
34141 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE111265 |
Dnmt1 binds and represses genomic retroelements via DNA methylation in mouse early embryos (RNA-seq) |
| GSE111266 |
Dnmt1 binds and represses genomic retroelements via DNA methylation in mouse early embryos |
|
| Relations |
| BioSample |
SAMN08623881 |
| SRA |
SRX3752125 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
