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Sample GSM2974952 Query DataSets for GSM2974952
Status Public on Sep 30, 2018
Title mNET_unph_rep2
Sample type SRA
Source name seedling
Organism Arabidopsis thaliana
Characteristics cultivar: Col-0
tissue: seedling
age: 12 days
Growth protocol The Arabidopsis (Col-0 ecotype) seeds were germinated and grown in 1/2 MS for 12 days at 20°C under 16 hour light/8 hour dark.
Extracted molecule total RNA
Extraction protocol The tissue powder was solubilized in 30 ml lysis buffer (50 mM HEPES, pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 5 mM 2-mercaptoethanol, 2 μg/ml pepstatin A, 2 μg/ml aprotinin, 1 mM PMSF), mixed well and filtrated through 110 μm nylon mesh and a 40 μm cell strainer. The nuclei were spanned down at 3,200 g for 20 min at 4°C, followed by two washes with HBB buffer (25 mM Tris–HCl, pH 7.6, 0.44 M sucrose, 10 mM MgCl2, 0.1% Triton-X, 10 mM 2-mercaptoethanol) and HBC buffer (20 mM Tris–HCl, pH 7.5, 352 mM sucrose, 8 mM MgCl2, 0.08% Triton-X, 8 mM 2-mercaptoethanol, 20% glycerol).
The obtained nuclei pellet were resuspended with 1 ml MNase buffer (20 mM Tris–HCl, pH 8.0, 5 mM NaCl, and 2.5 mM CaCl2) and digested with 20 U MNase (TaKaRa, 20U/μl) for 5 minutes at 37°C rotating at 1,400 rpm. Stopped the digestion by adding 40 μl 500 mM EDTA. Pol II-nascent RNA complex was released by mild sonication. Supernatants were collected by centrifuging at 16,000 g for 5 min at 4°C and Pol II complex were immunoprecipitated according to Nojima et al. [14]. The Pol II bond nascent RNAs were treated by T4 PNK on beads and extracted with 500μl TRIzol Reagent (Invitogen). The IP RNA was resolved on an 8% TBE-urea polyacrylamide gel and RNA with size from 35-100 nt was recovered. The 35-100 nt IP RNA was used to construct the NET-SEQ cDNA library with NEXTflex™ Small RNA-Seq Kit v3 (Bioo Scientific) according to the manual. The cDNA library was resolved in a non-denaturing 6% TBE polyacrylamide gel and size region 150-230 bp was recovered. cDNA library concentration was determined by Qubit QuantIT (Invitrogen) and kapa quantitative realtime PCR.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
Description nascent RNA
Data processing Library strategy: mNET-seq
Sequenced reads were trimmed for adaptor sequence, and masked for low-quality sequence, then mapped to TAIR10 whole genome using bowtie v1.1.2.
Genome_build: TAIR10
Supplementary_files_format_and_content: tab-delimited text files include the position information of RNA Pol II for each sample
Supplementary_files_format_and_content: bedgraph
Submission date Feb 01, 2018
Last update date Oct 31, 2018
Contact name Zhicheng Dong
Organization name Guangzhou University
Department School of Life Sciences
Street address 230 Wai Huan Xi Road,Guangzhou Higher Education Mega Center
City Guangzhou
State/province Guangdong
ZIP/Postal code 510006
Country China
Platform ID GPL19580
Series (1)
GSE109974 RNA polymerase II activity revealed by GRO-seq and pNET-seq in Arabidopsis
BioSample SAMN08450247
SRA SRX3638180

Supplementary file Size Download File type/resource
GSM2974952_mNET_unph_rep2.txt.gz 5.5 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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