GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM954718 Query DataSets for GSM954718
Status Public on Jun 30, 2015
Title RNA-seq_hlp1
Sample type SRA
Source name RNA-seq_hlp1
Organism Arabidopsis thaliana
Characteristics tissue: seedlings
tag: hlp1
Treatment protocol The seedlings were ground into fine power in liquid nitrogen and RNA was extracted by TRNzol reagent (Tiangen).
Growth protocol All of the Arabidopsis thaliana lines used in this study were in the Col background. Plant growth, flowering time analysis, and plant transformation were performed as reported previously (Pei et al., 2007).
Extracted molecule total RNA
Extraction protocol 12-day-old seedlings (from MS plates containing 3% sucrose grown under LD at 23°C) expressing either CFP-HLP1 or CFP-HLP1ΔRRM were soaked in ice-cold PBS buffer and irradiated twice for 400 mJ/cm2 in the Hoefer UVC 500 Ultraviolet Crosslinker (GE). CLIP experiment was performed as described (Licatalosi et al., 2008; Xue et al., 2009). Poly(A) site sequencing was performed as described (Fu et al., 2011; Shepard et al., 2011). Briefly, poly(A) RNAs were purified using the mRNA purification kit (Invitrogen), and heat fragmented at 95°C for 30 min. Reverse transcription (Superscript Ⅱ, Invitrogen) was performed using our modified HITS-3’ adaptor at 42°C for 30 min, then add the HITS-5’ adaptor (a SMART oligo) and incubated for additional 30 min. The cDNAs were purified using the Qiagen PCR Cleanup kit and the second strand cDNAs were synthesized by 3 cycles PCR using Phusion DNA polymerase (NEB) and the PE1.0 and PE2.0 primers. The PCR products were separated by 2% agarose gel and the 200-300 bp bands were excised and purified. Gel-extracted DNAs were further amplified by a 13-cycle PCR. The PCR products were purified using the Qiagen PCR Cleanup kit. TA-cloning was performed before Illumina GA Ⅱ sequencing.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer II
Data processing Two independent CLIP-seq libraries (wild type HLP1 and △RRM) were sequenced on Illumina GAⅡ. 3'-end adaptors and the 5'-end four barcodes were removed using FASTX toolkit ( Tags less than 14 nt in length or adapter-only were discarded. Tags ranging from 14~19 nt (no mismatch allowed) and tags ≥ 20 nt (two mismatches allowed) were aligned to the Arabidopsis genome (TAIR10) using bowtie (0.12.7) (Langmead et al., 2009). The identical sequences with the same 4 nt random barcodes were regarded as one tag to exclude PCR duplication. The RNA-seq libraries for Col, hlp1-1, atprmt10-1 and double mutants were constructed according to strand-specific mRNA-Seq Sample Preparation Protocol. All the raw RNA-seq reads were aligned to the Arabidopsis genome (TAIR10) by using TopHat (1.3.0) (Trapnell et al., 2009) with no more than six mismatches. PAS-seq tags less than 20 nt were discarded. All tags more than 50nt were trimmed to 50 nt and reversely complemented because the tags were sequenced from 3' end of transcripts (Fu et al., 2011). Then trimmed tags were mapped to the Arabidopsis genome (TAIR10) using bowtie (0.12.7) (Langmead et al., 2009), allowing 2 mismatches. Only unique mapped tags from CLIP-seq/RNA-seq/PAS-seq were kept for downstream analyses and were tranfered into wiggle files which can be displayed in IGV (Robinson et al., 2011).
Genome_build: TAIR10
Submission date Jul 02, 2012
Last update date May 15, 2019
Contact name xiaofeng cao
Phone 86-10-64869203
Organization name Institute of Genetics and Developmental Biology
Department State Key Laboratory of Plant Genomics
Street address West Lincui Road, Chaoyang District
City Beijing
ZIP/Postal code 100101
Country China
Platform ID GPL9302
Series (1)
GSE39051 Integrative genome-wide analysis reveals HLP1, a novel RNA-binding protein, regulates plant flowering by targeted alternative polyadenylation
SRA SRX157469
BioSample SAMN01085332

Supplementary file Size Download File type/resource
GSM954718_RNA-seq_hlp1.bed.gz 56.6 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap