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| Status |
Public on Sep 13, 2015 |
| Title |
41_RIP_input |
| Sample type |
SRA |
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| Source name |
flowers_41_RIP_input
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Columbia
genotype: dcl2/3/4
tissue: immature inflorescence
antibody: AGO4 antibodies (Agrisera #AS09 617)
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| Growth protocol |
The wild-type and mutant alleles in this study were in the background of Arabidopsis thaliana ecotype Columbia and have been previously described. Plants were grown in a growth chamber with 16 hour of light for five weeks. Immature inflorescence tissues including inflorescence meristem and early stages floral buds (up to stage 11/12) were collected.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was first treated with RiboMinus™ Plant Kit for RNA-Seq (Invitrogen A10838-08) to remove rRNA, followed by size selection of RNA on a 15% UREA TBE Polyacrylamide gel (Invitrogen, EC6885BOX). Gels containing RNA with size between 15- to 27-nt were kept for small RNA library, while gels containing 28- to ~300-nt RNA were kept for PATH library. ChIP-seq was performed as described previously (Johnson et al., 2014). 5 ug Pol II antibodies (Abcam #ab817) were used for each ChIP. Libraries for Pol II ChIP-seq were generated using the Ovation Ultralow DR Multiplex System (NuGen #0330) and sequenced at a length of 50 bp. ChIP-seq data was visualized using ngsplot (Shen et al., 2014). AGO4-RIP was performed as previously described (Ji et al., 2011) with commercial AGO4 antibody (Agrisera #AS09 617). RNA isolated from RIP was used for library construction with Illumina TruSeq RNA Sample Preparation Kit (RS-122-2001) according to the standard manual. For BS-seq, DNA was isolated using the DNeasy Plant Mini Kit (Qiagen #69104) according to manufacturer instructions and quantified using the Qubit dsDNA High Sensitivity Kit (Life Technologies #Q32851). Libraries were constructed with 30ng DNA using the Ovation Ultralow Methyl-Seq Library Systems (NuGEN #0335). Bisulfite conversion was done using the EpiTect Bisulfite Kit (Qiagen # 59104). BS-seq Libraries were sequenced at a length of 50 bp.
After gel elution, library construction for both sRNA and PATH was done using the Illumina TruSeq Small RNA Sample Preparation Kit (RS-200-0012), except that at the final size selection step, PCR products were separated on a 6% TBE Polyacrylamide gel (Invitrogen, EC6265BOX) and selected for the range from 120- to ~1000-bp for PATH library. Gel-eluted PCR products with different TruSeq index sequences were pooled and sent for Illumina sequencing. The mRNA library was constructed using the Illumina TruSeq RNA Sample Preparation Kit (RS-122-2001) according to the standard manual. PATH libraries were sequenced using either paired-end mode with length of read1 being 120-bp and length of read2 being 30-bp (PE120+30), or single-end 100-bp (SE100); while sRNA and mRNA libraries were sequenced with single-end 50-bp (SE50). All sequencing was carried out on HiSeq2500 machines at the Broad Stem Cell Research Center (BSCRC) sequencing core at University of California, Los Angeles.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
RIP
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| Data processing |
In general, qseq files received from the sequencing core were demultiplexed with an in-house Perl script and converted to fastq files for downstream analysis. For small RNA and PATH data, original reads were first trimmed using Cutadapt (v1.4), then mapped to the reference TAIR10 genome using Bowtie34 allowing only one unique hit (-m 1). We allowed zero mismatch for small RNA mapping (-v 0) and up to three mismatches for PATH mapping (-v 3). mRNA data were mapped using Tophat allowing two mismatches and only one unique hit.
Genome_build: TAIR10
Supplementary_files_format_and_content: ChIP-seq samples are associated with wig coverage files; For BS-seq processed data, the format is "position, strand, context(x=CG,y=CHG,z=CHH), methylated count, unmethylated count", RIP-seq samples and small RNA-seq samples are associated with files containing the sequence and raw read count.
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| Submission date |
Aug 11, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Steve Jacobsen |
| Organization name |
University of California, Los Angeles
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| Department |
Department of Molecular, Cell and Developmental Biology
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| Street address |
4045 Terasaki Life Sciences Building
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
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| Platform ID |
GPL17639 |
| Series (1) |
| GSE61439 |
Pol-IV dependent transcripts in Arabidopsis |
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| Relations |
| BioSample |
SAMN03981866 |
| SRA |
SRX1142683 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1848711_tag_count_RIP_input.txt.gz |
7.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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