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| Status |
Public on Mar 13, 2020 |
| Title |
Microsome polysomal RNA ddm1 Rep 3 |
| Sample type |
SRA |
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| Source name |
Inflorescence tissues
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
genotype: ddm1-2
expressing: 35S:FLAG-RPL18
tissue: Inflorescence tissues
fraction: Microsome polysomal RNA
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| Growth protocol |
Plants were grown under long day conditions at 22 °C. Seeds were grown on ½ Murashige and Skoog (MS) medium and seedlings were transplanted to soil 7 day after germination.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Microsomes were isolated using sucrose-gradient ultracentrifugation. Polysomal RNA was extracted by FLAG-RPL18 immunoprecipitation.
ScriptSeq v2 RNA-Seq Library Preparation Kit
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
RNA-seq: FASTQ files were processed for adaptor trimming using cutadapt. Tophat v2.0.12 was used for mapping. Cuffnorm was used to obtain FPKM values from bam files.
Small RNA-seq: After trimming the 3’ adapter and filtering collapsed reads according to length and quality, filtered reads were mapped with bowtie reporting all multi-mappers. Only perfect match reads were used for down-stream analysis, and reads mapped to multiple genomic locations where normalized by dividing non-redundant read counts by the number of genomic hits, and subsequently calculating the number of reads per million of filtered (18-30nt) and perfectly mapped reads.
Short read VLP DNA-seq: Adapters were trimmed from raw reads with Skewer in paired-end mode and read pairs with both mates longer than 25 nt were retained.
Short read VLP DNA-seq: Reads were aligned to the TAIR10 genome with STAR in two-pass mode to improve spliced alignment at unannotated introns. Reads mapping equally well to multiple locations were randomly assigned, and chimeric/split read alignments were output separately from concordant alignments. Optical and PCR duplicates were removed from the alignments with picard-tools (http://broadinstitute.github.io/picard). Genome-wide coverage was calculated with bamCoverage from deepTools and normalized to reads per million mapped. Pairwise differential expression at TAIR10 transposon loci was tested across three wild-type, two ddm1, and three ddm1rdr6 replicates using quasi-likelihood F-tests in edgeR, controlling FDR at 5% and a log2(fold-change) threshold of 2.
Long read VLP DNA-seq: Basecalling was performed offline with Guppy v2.3.1 using the default model. Using porechop (https://github.com/rrwick/Porechop), ONT sequencing adapters were trimmed from 5’ ends, 3’ ends, and the middle of raw reads. Reads with middle adapters were split.
Long read VLP DNA-seq: Remaining reads longer than 100 bp were aligned to the TAIR10 reference with minimap2 for coverage and read alignment plots. Structural variants were called on NGMLR alignments using Sniffles with default parameters, except minimum read support was reduced to 3. RPM-normalized coverage was calculated with bamCoverage from deepTools and normalized to reads per million mapped.
Genome_build: TAIR10
Supplementary_files_format_and_content: RNA-seq: Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values are shown for each gene identification (ID)
Supplementary_files_format_and_content: Small RNA-seq: txt files with 18-30nt reads and counts, and RPM of 20-24nt reads mapped to TAIR10 features
Supplementary_files_format_and_content: Short read VLP DNA-seq: bigWig
Supplementary_files_format_and_content: Long read VLP DNA-seq: bigWig, vcf (Structural variant calls produced by Sniffles on nanopore long-read alignments to the TAIR10 genome. The number of reads supporting each SV call is given in the RE tag.)
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| Submission date |
Mar 27, 2019 |
| Last update date |
Mar 13, 2020 |
| Contact name |
Robert A Martienssen |
| E-mail(s) |
martiens@cshl.edu
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| Organization name |
Cold Spring Harbor Laboratory
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| Department |
Delbruck Bldg.
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| Lab |
Martienssen
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| Street address |
1 Bungtown Rd
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| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
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| Platform ID |
GPL13222 |
| Series (1) |
| GSE128932 |
Arabidopsis retrotransposon virus-like-particles and their regulation by epigenetically activated small RNA |
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| Relations |
| BioSample |
SAMN11265732 |
| SRA |
SRX5581851 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3688582_Mpoly_ddm1_3.txt.gz |
186.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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