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Status |
Public on Oct 27, 2019 |
Title |
ADAR1 WT ADAR2 KO Adult Brain - Whole. Replicate 1 (502) A2KOr1 |
Sample type |
SRA |
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Source name |
ADAR1 WT ADAR2 KO 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: -/- gria2 genotype: R/R ifih1 genotype: -/- tissue: Adult Brain replicate: 1
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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 |
HiSeq X Ten |
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Description |
ADAR1 WT ADAR2 KO Adult Brain - Whole. Replicate 1 (502) Adar2KO_502 S1_Adar1_+_+_Ifih_-_-_Adar2_-_-_Gria2_RR
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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 |
GPL21273 |
Series (1) |
GSE132214 |
The majority of A-to-I RNA editing is not required for mammalian homeostasis |
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Relations |
BioSample |
SAMN11960673 |
SRA |
SRX5974605 |
Supplementary file |
Size |
Download |
File type/resource |
GSM3854159.jacusa.known.txt.gz |
279.0 Kb |
(ftp)(http) |
TXT |
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Raw data are available in SRA |
Processed data are available on Series record |
Processed data provided as supplementary file |
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