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Sample GSM277608 Query DataSets for GSM277608
Status Public on Apr 21, 2008
Title Col-0_smRNA-seq
Sample type SRA
Source name Immature floral tissue
Organism Arabidopsis thaliana
Characteristics Columbia-0, unopened flower buds
Treatment protocol Immature (unopened) flower buds were collected and frozen in liquid nitrogen.
Growth protocol All plants were grown in potting soil (Metro Mix 250; Grace-Sierra, Boca Raton, FL) at 23C under a 16-hour light/8-hour dark cycle.
Extracted molecule total RNA
Extraction protocol smRNA-seq library construction protocol:
Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Immediately following RNA precipitation, the flow through from the anion-exchange chromatography column was further precipitated in another 2.5 volumes of 100% ethanol (smRNA fraction). The smRNA fraction was further purified by a phenol-chloroform extraction and an additional ethanol precipitation. Small RNAs were resolved by electrophoresis of 2.5 mg of the smRNA fraction and 7.5 mg of total RNA on 15% polyacrylamide gels containing 7 M urea in TBE buffer (45 mM Tris-borate, pH 8.0, and 1.0 mM EDTA). A gel slice containing RNAs of 15 to 35 nucleotides (based on the 10 base pair ladder size standard (Invitrogen, Carlsbad, CA)) was excised and eluted in 0.3 M NaCl rotating at room temperature for 4 hours. The eluted RNAs were precipitated using ethanol and resuspended in diethyl pyrocarbonatetreated deionized water. Gel-purified smRNA molecules were ligated sequentially to 5' and 3' RNA oligonucleotideadapters using T4 RNA ligase (10 units/mL) (Promega, Madison, WI). The 5' RNA adapter (5' - GUUCAGAGUUCUACAGUCCGACGAUC - 3') possessed 5' and 3' hydroxyl groups. The 3' RNA adapter (5'-pUCGUAUGCCGUCUUCUGCUUGidT-3') possessed a 5' mono-phosphate and a 3' inverted deoxythymidine (idT). The smRNAs were first ligated to the 5' RNA adapter. The ligation products were gel eluted and ligated to the 3' RNA adapter as described above. The final ligation products were then used as templates in a reverse transcription (RT) reaction using the RT-primer (5' - CAAGCAGAAGACGGCATACGA - 3') and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). This was followed by a limited (15 cycle) PCR amplification step using the PCR reverse (5'-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3') and forward (5'-CAAGCAGAAGACGGCATACGA-3') primers and Phusion hot-start high fidelity DNA polymerase (New England Biolabs, Cambridge, MA). All oligonucleotides were provided by Illumina (Illumina, San Diego, CA). The amplification products were separated by electrophoresis on a 6% polyacrylamide gel in TBE buffer, eluted in 0.3 M NaCl rotating at room temperature for 4 hours, precipitated using ethanol, and resuspended in nuclease-free water
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina Genome Analyzer
Description Sequence information was extracted from the image files with the Illumina Firecrest and Bustard applications and mapped to the Arabidopsis (Col-0) reference genome sequence (TAIR 7) with the Illumina ELAND algorithm. ELAND aligns 32 bases or shorter reads, allowing up to two mismatches to the reference sequence. For reads longer than 32 bases, only the first 32 bases will be used for alignment, while the remaining sequence will be appended regardless of similarity to the reference sequence. A Perl script was used to truncate the appended sequence at the point where the next four bases contain two or more errors relative to the reference sequence.
Data processing Prior to alignment of the smRNA reads, a custom Perl script was used to identify the first seven bases of the 3? adapter sequence, and the read was truncated up to the junction with the adaptor sequence. Each of the reads was then mapped to the genome with BLAST using a word size of 10 and expectation value of 10. Only perfect matches were accepted, as these shorter reads will have a higher tendency to falsely map than longer reads. No further analysis was performed on reads that do not contain the adapter sequence, as their size class could not be determined precisely.
Submission date Mar 26, 2008
Last update date May 15, 2019
Contact name Joseph R Ecker
Phone 8584534100
Organization name HHMI-Salk-Institute
Department Genomic Analysis Laboratory
Lab Ecker lab
Street address 10010 North Torrey Pines Road
City La Jolla
State/province CA
ZIP/Postal code 92037
Country USA
Platform ID GPL9062
Series (2)
GSE10877 Highly integrated single base resolution maps of the epigenome in Arabidopsis
GSE10967 Highly integrated epigenome maps in Arabidopsis - small RNA sequencing
SRA SRX002508
BioSample SAMN02049271

Supplementary file Size Download File type/resource
GSM277608.txt.gz 27.1 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data not provided for this record
Raw data are available in SRA

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