|
| Status |
Public on Jul 15, 2018 |
| Title |
Small RNA-seq from Mbd3f/- system day1 |
| Sample type |
SRA |
| |
|
| Source name |
Mbd3f/- cell line, Day1 post DOX induction
|
| Organism |
Mus musculus |
| Characteristics |
genotype/cell type: Mbd3f/- cell line
stage of reprogramming: Day1 post DOX induction
|
| Treatment protocol |
Reprogramming dynamics samples were generated by adding Dox to Secondary MEF. ES and iPSC samples were harvested after 3-4 days of growth in N2B27 2i\LIF medium. Reprogramming samples of days 3-8, ES and iPSC samples were plated on irradiated human foreskin fibroblasts (HFF).
|
| Growth protocol |
Maintenance and reprogramming (Day 3 and beyond) of murine naïve pluripotent cells were conducted in bovine serum-free N2B27-based media or KSR media: N2B27- based media: 500ml KO-DMEM (Invitrogen), 5ml N2 supplement (Invitrogen; 17502048), 5ml B27 supplement (Invitrogen; 17504044), 15% knockout serum replacement (Invitrogen – 10828), 1mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), penicillin-streptomycin (Invitrogen), 5mg/ml BSA (Sigma). KSR media: 500 ml DMEM (Invitrogen) , 1mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), penicillin- streptomycin (Invitrogen). Naïve conditions for murine PSCs included 10μg recombinant human LIF (Millipore; LIF1005). Where indicated 2i was added 48 hours after OSKM induction: small-molecule inhibitors CHIR99021 (CH, 3μM- Axon Medchem) and PD0325901 (PD 0.2 or 1μM - Axon Medchem).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1ug of total RNA from each sample was processed using the Truseq small RNA sample preparation kit (RS-200-0012 Illumina) followed by 12 cycles of PCR amplification. Libraries were evaluated by Qubit and TapeStation. For purification of the small RNA fragments, they were size selected using Blupippne machine (Sage Science) with 3% gel cassette followed by clean-up with minielute PCR purification kit (Qiagen). The libraries were constructed with different barcodes to allow multiplexing of 11 samples.
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
We used Illumina CASAVA 1.8.2 software to generate fastq files.
Bowtie software version 2 was used to align reads to mouse mm10 reference genome (UCSC, December 2011).
FPKM values were calculated over all genes in mm10 assembly GTF (UCSC, December 2011), using cufflinks version 2.2.1.
Genes annotated as rRNA, miRNA, snoRNA were selected for further analysis.
Genome_build: mm10
|
| |
|
| Submission date |
Jul 03, 2018 |
| Last update date |
Jul 15, 2018 |
| Contact name |
Noa Novershtern |
| E-mail(s) |
noa.novershtern@weizmann.ac.il
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Molecular Genetics
|
| Street address |
Weizmann Institute
|
| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE102518 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems |
| GSE116573 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems [smallRNA-Seq] |
|
| Relations |
| BioSample |
SAMN09537157 |
| SRA |
SRX4337031 |
