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Sample GSM3688571 Query DataSets for GSM3688571
Status Public on Mar 13, 2020
Title Total RNA ddm1rdr6 Rep 1
Sample type SRA
 
Source name Inflorescence tissues
Organism Arabidopsis thaliana
Characteristics ecotype: Col-0
genotype: ddm1-2 rdr6-15
expressing: 35S:FLAG-RPL18
tissue: Inflorescence tissues
fraction: Total RNA
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.
Extracted molecule total RNA
Extraction protocol Polysomes of FLAG-RPL18 were immunoprecipitated with anti-FLAG. Input RNA was set aside for total RNA. Eluted polysome-associated RNA was extracted using Direct-zol RNA miniprep kit.
ScriptSeq v2 RNA-Seq Library Preparation Kit
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
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.)
 
Submission date Mar 27, 2019
Last update date Mar 13, 2020
Contact name Robert A Martienssen
E-mail(s) martiens@cshl.edu
Organization name Cold Spring Harbor Laboratory
Department Delbruck Bldg.
Lab Martienssen
Street address 1 Bungtown Rd
City Cold Spring Harbor
State/province NY
ZIP/Postal code 11724
Country USA
 
Platform ID GPL13222
Series (1)
GSE128932 Arabidopsis retrotransposon virus-like-particles and their regulation by epigenetically activated small RNA
Relations
BioSample SAMN11265743
SRA SRX5581840

Supplementary file Size Download File type/resource
GSM3688571_total_ddm1rdr6_1.txt.gz 181.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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