|
| Status |
Public on Dec 09, 2016 |
| Title |
ESC RNA CN 2 |
| Sample type |
SRA |
| |
|
| Source name |
poly(A)RNA
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129S2/SvPasCrl
Sex: female
age: --
tissue: embryonic stem cells
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was isolated in biological triplicates from pulverized whole brain tissue and mouse embryonic stem cells (CN) as well as from nuclei (N) of both sources using TRIzol (Sigma Aldrich) following the manufacturer’s recommendations. RNA was treated with 2U of DNase I (NEB) for 15 min at 37°C and purified using RNA Clean & Concentrator 25 Kit (Zymo Research). Isolated RNA was then subjected to two rounds of poly(A)RNA enrichment using fresh Dynabeads (Ambion) for each round.
Sequencing libraries were prepared using the ScriptSeq V2 RNA-Seq Library Preparation Kit (Epicentre), purified with AMPure XP beads (Beckman Coulter) and quantified using KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems). RNA fragmenting was omitted as bisulfite treatment results in fragmentation to 100-250 nt. Libraries were multiplexed at 11 pM and sequenced on an Illuma HiSeq 1500 plattform using 100 bp single end reads
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
transcriptomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
raw fastq files filtered (fastq_quality_filter -p 85 -q 30) and quality trimmed (fastq_quality_trimmer -t 30 -l 35) using fastx_tookit (version 0.0.14), adapter sequences were removed using flexbar (version 2.5)
clean reads were aligned to the GRCm38/mm10 genome using meRanGs available with meRanTK (version 1.0) using default settings
m5C candidate sites were called using meRanCall available with meRanTK (version 1.0) (Rieder et al. 2016) with FDR < 0.01. Based on the m-bias plots obtained from meRanGs, 10 bases on the 5’ end of forward- and 7 bases on the 5’ end of reverse reads were excluded from methylation calling. Furthermore, only bases with a base-call quality score of Q >= 35 for single end- and Q >= 30 for paired-end reads were considered for methylation calling. Candidate cytosine positions were covered by at least 10 reads and had a conversion rate less than 0.8.
An m5C candidate had to be present in all three replicates of a given sample. Subsequently, the full length transcripts containing an m5C candidate were extracted from the RefSeq database (GRCm38.p3) and subjected to secondary structure analysis using the RNAfold of the Vienna RNA package (version 2.2.8) (Lorenz R et al. 2011). We calculated the maximum expected accuracy (MEA) structure at 70°C using a gamma of 0.1. The maximum allowed distance between two bases in a pair was set to 150 nt. For introns and non-annotated sequences only 300 nt around the m5C candidate position were subjected to folding analysis. Only candidate m5C sites that were predicted not to be base-paired in the resulting structure were retained
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content:
Excel (.xlsx) files containing methylation sites found in minimum 3 replicates - additionally, excel files with information about m5C sites in individual replicates
Files containing m5C sites in minimum 3 replicates (Brain_total; Brain_nuclear; ESC_total; ESC_nuclear) - files containing m5C sites in the individual replicates (Brain nuclear_indReplicates; Brain total_indReplicates; ESC nuclear_indReplicates; ESC total_indReplicates)
|
| |
|
| Submission date |
Jun 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexandra Lusser |
| E-mail(s) |
Alexandra.Lusser@i-med.ac.at
|
| Phone |
+43 512 9003-70210
|
| Organization name |
Biocenter Medical University Innsbruck
|
| Department |
Molecular Biology
|
| Street address |
Innrain 80
|
| City |
Innsbruck |
| State/province |
Tyrol |
| ZIP/Postal code |
6020 |
| Country |
Austria |
| |
|
| Platform ID |
GPL18480 |
| Series (1) |
| GSE83432 |
Distinct 5-methylcytosine profiles in poly(A)RNA from mouse embryonic stem cells and the brain |
|
| Relations |
| BioSample |
SAMN05255753 |
| SRA |
SRX1850434 |
