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| Status |
Public on Oct 27, 2019 |
| Title |
ADAR1 WT MDA5 knockout Adult Brain - Whole. Replicate 2 (82B) WTr2 [re-analysis] |
| Sample type |
SRA |
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| Source name |
ADAR1 WT MDA5 knockout Adult Brain
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
age: 12 week old; adult
Sex: male
adar1 genotype: WT
adar2 genotype: WT
gria2 genotype: WT
ifih1 genotype: -/-
tissue: Adult Brain
replicate: 2
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| Growth protocol |
RNA was harvested from Single cell suspensions of whole feotal brain using Trizol reagent . >2.5 ug of total RNA was used for the construction of sequencing libraries.
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| Extracted molecule |
total RNA |
| Extraction protocol |
[75bp] Post ribosome depleted RNA was purified and subjected to indexing and library preparation using the Kapa Stranded RNA-seq Library Preparation Kit for Illumina Platforms (Kapa Biosystems). Library amplification was performed for 9 cycles of PCR. The resultant libraries were pooled and sequenced on a single run using the Illumina NextSeq500 with 75bp paired end reads at the Ramaciotti Centre for Genomics (UNSW, Australia).
[150bp] Total RNA was isolated from the whole brain from 12-week-old males Adar1E861A/+Ifih1-/-Adar2+/-Gria2R/R (dHet), Adar1+/+Ifih1-/-Adar2-/-Gria2R/R (Adar2-/-) and Adar1E861A/E861AIfih1-/- Adar2-/-Gria2R/R (Quad) mice (n= 3/genotype). Whole brain was lysed in Trisure and purified using DirectZol columns. Post ribosome-depleted RNA was purified and subjected to indexing and library preparation using the Kapa Stranded RNA-seq Library Preparation Kit (Kapa Biosystems) and sequenced at PE150bp on the Illumina platform by the Novogene (Hong Kong).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
This is duplicated sample record of GSM2474528 for the convenient retrieval of the complete raw data from SRA
ADAR1 WT MDA5 knockout Adult Brain - Whole. Replicate 1 (82B)
MDA5_KO_82B
ADAR_DKO_ADARwt_MDA5ko_RAdar82B
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| Data processing |
Illumina Casava1.7 software used for basecalling.
Preprocessing: Sequenced reads (150bp) were trimmed for adaptor sequence and low quality reads using fastp (v 0.19.5, Shifu Chen, fastp: A fast FASTQ preprocessor with full features, (2017), GitHub repository, https://github.com/OpenGene/fastp ). Command line used: fastp --trim_front1 10 --trim_front2 10
Preprocessing: Sequenced reads (75bp) from GSE94387 were trimmed for adaptor sequence and low quality reads using fastp (v 0.19.5, Shifu Chen, fastp: A fast FASTQ preprocessor with full features, (2017), GitHub repository, https://github.com/OpenGene/fastp ). Command line used: fastp --trim_front1 10 --trim_front2 10 --trim_tail1 1 --trim_tail2 1
Preprocessing: Reads mapping to rRNA were removed using Bbmap . Command line used: bbsplit.sh minratio=0.56 minhits=1 maxindel=16000
RNA Editing: Editing calling of known sites (RNA vs mm10) was performed using JACUSA 2.0.0-RC540 (https://github.com/dieterich-lab/JACUSA): parameters used: -F 1024 -filterNH_ 99, -filterNM_ 99, -c 3 -P RF-FIRSTSTRAND.
RNA Editing: Calling of differential editing in known sites (RNA genotype vs RNA genotype) was performed using JACUSA 2.0.0-RC540. Briefly, call-2 was used to determine the difference in editing level for all known sites (all replicates of genotype A vs all replicates of genotype B). Duplicate reads were removed.
Gene expression: Trimmeds reads were aligned using Salmon (version v0.11.3) against mm10 (Annotation: gencode.mm10.vM14.annotation.gtf).
Gene expression: Data was normalised in Degust (http://victorian-bioinformatics-consortium.github.io/degust/) using EdgeR (McCarthy, J. D, Chen, Yunshun, Smyth and K. G (2012). “Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation.” Nucleic Acids Research, 40(10), pp. -9.). Genes with count >1 and CPM > 1 in at least 3/3 samples of a given genotype were considered for further analysis. Each comparison (E861A vs WT, A2KO vs Het, DKO vs Het) was performed seperately. Differential expression was performed in Degust using EdgeR.
Genome_build: mm10
Supplementary_files_format_and_content: count
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| Submission date |
Jun 05, 2019 |
| Last update date |
Oct 29, 2019 |
| Contact name |
Alistair Morgan Chalk |
| E-mail(s) |
achalk@svi.edu.au, alistair.chalk@gmail.com
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| Organization name |
St Vincent's Institute of Medical Research
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| Department |
Stem Cell Regulation Unit
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| Lab |
Walkley
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| Street address |
9 Princes st, Fitzroy
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| City |
Melbourne |
| State/province |
VIC |
| ZIP/Postal code |
3065 |
| Country |
Australia |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE132214 |
The majority of A-to-I RNA editing is not required for mammalian homeostasis |
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| Relations |
| Reanalysis of |
GSM2474528 |
| BioSample |
SAMN11960664 |
| SRA |
SRX5974612 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3854166.jacusa.known.txt.gz |
265.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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