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| Status |
Public on Mar 21, 2022 |
| Title |
1.-Esrrb_miRNA_ Day 0 |
| Sample type |
SRA |
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| Source name |
Mouse ES_AINV15
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| Organism |
Mus musculus |
| Characteristics |
sample type: Esrrb_ Recue clone
treatment: +Dox
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| Treatment protocol |
Esrrb_R rescue clone was grown in +Dox (1ug/ml) media_Day0; Dox withdrawal was performed for 5 days performing miRNA-Seq at the respective days: Day0 ( Esrrb expressed) and days 1,3 and 5 ( Esrrb non expressed)
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| Growth protocol |
For all experiments,Esrrb_R clone was grown on irradiated MEFS in mESC media ( D-MEM–High Glucose (Dulbecco’s modified Eagle’s medium-1X-High Glucose) (Gibco®, Invitrogen), 15% FBS (Fetal bovine serum) (Hyclone, Thermo Scientific), 100 mM MEM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM L-glutamine, Penicillin/Streptomycin (Gibco, Invitrogen) and 103 U/ml LIF (Chemicon, Millipore). All cell cultures were maintained at 37ºC with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Short transcript libraries were generated using size selected RNA. Total RNA at different time points after downregulation of Esrrb was extracted with Qiazol® (Qiagen) with subsequent enrichment for small RNAs (<200 bp) using the miRNeasy® Mini purification kit according to the manufacturers’ instructions for total RNA isolation that includes the small fraction. Extracted RNA (40 µg) was combined with trace amounts of 5´-32P-labeled RNA standards, 19-nt 5’-CGUACGCGGGUUUAAACGA-3’ and 24-nt 5’-CGUACGCGGAAUAGUUUAAACUGU-3'. RNA was then fractionated on a 15% polyacrylamide, 8 M urea gel (Bio-Rad). A gel fragment spanning both the 18 nt and 24-26 nt internal standards was excised, and RNA was eluted and ethanol-precipitated in siliconized tubes, with 20 µg of glycogen as carrier. Gel-purified 18-26 nt RNA was incubated with 50 µM pre-adenylylated 3´-adaptor oligonucleotide (Modban), 10X ATP-free ligase Buffer (500 mM Tri-HCl (pH=7.5-7.6), 100 mM MgCl2, 100mM DTT, 600 µg/ml BSA), and T4 RNA Ligase 2, truncated (New England BioLabs) in a 20 µl reaction at 37°C for 1 hour. The T4 RNA ligase reaction was purified on a 15% polyacrylamide, 8 M urea gel (Bio-Rad) by using the ligated forms of the standards as a guide for band excision. RNAs ligated with 3´-adaptor oligonucleotide was eluted and ethanol-precipitated in siliconized tubes, with 20 µg of glycogen. Ligated RNA product was used in a second T4 RNA ligase reaction for the 5’-adaptor oligonucleotide (Solexa Linker). Ligated products were gel-purified, excising the gel fragment spanning the doubly ligated standards. Gel-purified doubly ligated RNA was used in a standard 20µl RT reaction (SuperScript III, Invitrogen) with the RT primer, ATTGATGGTGCCTACAG (BanOne). The cDNA was PCR amplified with the 5’and 3’primers, generating products with an extended 3’ adaptor sequence. PCR products were digested with Pme I (NEB) at 37ºC for 3 hours. DNA fragments ranging from 108-113 bp were isolated from a low-melting point agarose gel. After further phenol extraction and ethanol precipitation, DNA samples were resuspended in Elution buffer (10 mM Tris/0.1% Tween) and then used according to the standard Solexa sequencing protocol (Illumina). Each library was run on one lane of the Solexa sequencer at the Genomic Sequencing Core at Icahn School of Medicine at Mount Sinai.
miRNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Day 0 (+D0X) Esrrb expressed
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| Data processing |
none provided by the submitter
Genome_build: mm9
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| Submission date |
Nov 27, 2021 |
| Last update date |
Mar 21, 2022 |
| Contact name |
Ana Sevilla |
| E-mail(s) |
anasevilla7@gmail.com
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| Phone |
675348398
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| Organization name |
Universidad de Barcelona
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| Street address |
Avda Diagonal 643. Edificio Malagef 4rd Floor, Faculty of Biology
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| City |
Barcelona |
| State/province |
ESP |
| ZIP/Postal code |
08028 |
| Country |
Spain |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE189678 |
Esrrb Regulates Specific Feed-Forward Loops to Transit from Pluripotency into Early Stages of Differentiation (RNA-Seq) |
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| Relations |
| BioSample |
SAMN23470449 |
| SRA |
SRX13242433 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5705934_Esrrb_PD_6_miRNA_Seq_Fafterblat_Esrrb_Day0_perfect_match.xlsx |
26.4 Mb |
(ftp)(http) |
XLSX |
| GSM5705934_Esrrb_PD_6_miRNAs_Seq_CCC_Galaxy34-_Day0-s_3_sequence.txt_collapsed_.fasta.gz |
2.6 Mb |
(ftp)(http) |
FASTA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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