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Sample GSM1668898

Query DataSets for GSM1668898

Status

Public on Sep 13, 2015

Title

16_sRNA_dcl2/3/4_#1

Sample type

SRA

Source name

flowers_16_sRNA_dcl2/3/4_#1

Organism

Arabidopsis thaliana

Characteristics

ecotype: Columbia
genotype: dcl2/3/4
tissue: immature inflorescence

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.

Extracted molecule

total RNA

Extraction protocol

not provided
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. 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.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Description

small RNA
library id: 16

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: Five libraries (1, 2, 15, 16, 29) were analyzed in-depth for ~7000 loci. The raw read counts at these loci for these five libraries are contained in the file "P4RNA_siRNA_and_mRNA_abundance_at_Pol-IV_loci.xlsx" available on the series record. The remaining libraries were analyzed at the level of "total abundance" at the same set of ~7000 loci. The number of "total abundance" for each library is listed in the in column H of the file "Summary_of_libraries.xlsx" which is also available on the series record.

Submission date

Apr 28, 2015

Last update date

May 15, 2019

Contact name

Steve Jacobsen

Organization name

University of California, Los Angeles

Department

Department of Molecular, Cell and Developmental Biology

Street address

4045 Terasaki Life Sciences Building