|
| Status |
Public on Feb 14, 2018 |
| Title |
ox+ 1:100,000 AthHEN1_S2-S+mES (replica 2) |
| Sample type |
SRA |
| |
|
| Source name |
Drosophila S2 cells + mouse embryonic stem cells
|
| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
treatment: Periodate-mediated oxidation
genotype: DmHen1-KO; FLAG-MYC-AthHEN1
cell type: Cultured Cells
|
| Treatment protocol |
50µg of total RNA (sum of Dme and Mmu total RNA) was used for the input and each dilution . Total RNA from mouse ES cells was mixed with decreasing amounts of total RNA from Drosophila S2 Hen1KO cells expressing AthHEN1, in the following ratios [Dme:Mmu]= 1:1; 1:10; 1:100; 1:1,000; 1:10,000; 1:100,000 (10 fold dilution of S2 total RNA). Each sample was split into two: one was subjected to treatment with sodium periodate (oxidised, +ox), the other was left untreated (unoxidised, -ox).
|
| Growth protocol |
Mouse embryonic stem (mES) cells (clone AN3-12) were cultured in 15 % FBS (Gibco), 1x Penicillin-Streptomycin solution (100 U/ml Penicillin, 0.1 mg/ml Streptomycin, Sigma), 2 mM L-Glutamine (Sigma), 1x MEM Non-essential amino acid solution (Sigma), 1 mM sodium pyruvate (Sigma), 50 µM 2-Mercaptoethanol (Gibco) and 20 ng/ml LIF (in-house produced). Cells were maintained at 37°C with 5% CO2 and passaged every second day. Drosophila S2 cells were grown in Schneider’s medium (Invitrogen) supplemented with 10% (v/v) FBS (Sigma) at 28°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cell pellets in Trizol using a phenol-chloroform-based standard protocol. Small RNA sequencing libraries were constructed from 50 µg of total RNA (sum of Mmu RNA + Dme RNA; the samples were then split into two: 25µg RNA/library ox- and 25 µg RNA/library ox+)
Small RNA libraries were constructed and sequenced as described in Ameres et al. (2010) with some modifications. 18-30 nt long RNAs for were size selected. Subsequent 2SrRNA depletion was performed with Dynabeads MyOne streptavidin C1 beads (Invitrogen) as described in Seitz et al. (2008). Small RNAs were ligated to 3’ and 5’ barcode adapters containing 4 random nucleotides at their ends to minimize ligation bias (Jayaprakash et al., 2011). Subsequent reverse transcription was performed with SuperScript III Reverse Transcriptase (Invitrogen) and cDNA samples were PCR amplified with the KAPA Real-Time Library Amplification Kit (Peqlab). Amplified cDNA was agarose gel purified and sequenced in an Illumina HiSeq2000 instrument.
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
18-30 nt size selection for miRNA seq
57674
|
| Data processing |
Basecalling RTA 1.18.64.0
The adapter (AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG) was cut once with cutadapt (v1.2.1).
The random 4mers on 5' and 3' were removed with fastx_trimmer of the fastx-toolkit (v0.0.13). The processed reads were filtered in respect of their size selection (18-30nt).
All reads were aligned first to M.musculus (mm10) genome with tailor (v1.1). The minimum length of exact match was 18.
Remaining unaligned reads were aligned to D.melanogaster (dm3) genome with tailor (v1.1). The minimum length of exact match was 18.
Reads were assigned to miRNA arms with htseq-count (v0.6.1p1). The parameters were stranded=yes, “intersection-nonempty” and a SAM file was the output. GTFs containing pre-miRNAs annotations (mirBase) of mouse and fly was used. The annotation was split in two halves assigned to 5' an and 3' of each miRNA region, respectively. Assigned reads (SAM file) were summed up for each miRNA half. Multimapping reads were counted only as fraction (1/number of mapped locations).
Genome_build: mm10 & dm3
Supplementary_files_format_and_content: count data of mouse and fly miRNAs in one tab separated text file
|
| |
|
| Submission date |
Nov 27, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Chiara Alberti |
| E-mail(s) |
chiara.alberti@imp.ac.at
|
| Organization name |
IMP
|
| Street address |
Campus-Vienna-Biocenter 1
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL21227 |
| Series (1) |
| GSE104466 |
Cell-type specific sequencing of microRNAs from complex animal tissues II |
|
| Relations |
| BioSample |
SAMN08102309 |
| SRA |
SRX3424833 |
