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| Status |
Public on Jun 30, 2015 |
| Title |
RNA-seq_col |
| Sample type |
SRA |
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| Source name |
RNA-seq_col
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: seedlings
tag: col
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| Treatment protocol |
The seedlings were ground into fine power in liquid nitrogen and RNA was extracted by TRNzol reagent (Tiangen).
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| 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).
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| 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| 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 (http://hannonlab.cshl.edu/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
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| Submission date |
Jul 02, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
xiaofeng cao |
| E-mail(s) |
xfcao@genetics.ac.cn
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| Phone |
86-10-64869203
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| Organization name |
Institute of Genetics and Developmental Biology
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| Department |
State Key Laboratory of Plant Genomics
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| Street address |
West Lincui Road, Chaoyang District
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| City |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
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| Platform ID |
GPL9302 |
| Series (1) |
| GSE39051 |
Integrative genome-wide analysis reveals HLP1, a novel RNA-binding protein， regulates plant flowering by targeted alternative polyadenylation |
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| Relations |
| SRA |
SRX157468 |
| BioSample |
SAMN01085331 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM954717_RNA-seq_Col.bed.gz |
48.3 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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