|
| Status |
Public on Jun 30, 2015 |
| Title |
PAS-seq_hlp1_mutant_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
PAS-seq_hlp1_mutant
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
background: col
genotype/variation: hlp1 mutant
tissue: seedlings
|
| 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). The atprmt10-1 (Niu et al., 2007) and flc-3 (Michaels and Amasino, 1999) mutants were described previously. The hlp1-1 mutant was isolated from the SALK collection (SALK_021452). Seedlings were grown on Murashige and Skoog (MS) plates containing 3% sucrose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The seedlings were ground into fine power in liquid nitrogen and RNA was extracted by TRNzol reagent (Tiangen).
Poly(A) site sequencing was performed as described with modifications at the HITS-3’ adaptor and sequencing primer (Fu et al., 2011; Shepard et al., 2011). Briefly, poly(A) RNAs were purified using a mRNA purification kit (Invitrogen), and heat fragmented at 95°C for 30 min. Reverse transcription (Superscript, Invitrogen) was carried out using our modified HITS-3’ adaptor at 42°C for 30 min, then the HITS-5’ adaptor (a SMART oligo) was added and incubated for an additional 30 min. The cDNAs were purified using a Qiagen PCR Cleanup kit and second strand cDNAs were synthesized by 3 cycles of PCR using Phusion DNA polymerase (NEB) and the PE1.0 and PE2.0 primers. The PCR products were separated on a 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 a Qiagen PCR Cleanup kit.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
high-throughput profiling of polyadenylation in hlp1 (mutant)
|
| Data processing |
library strategy: PAS-seq
Tags less than 20 nt after removal of the 3’ linker and 4 nt random barcodes were discarded. All tags were reverse-complemented because the tags were sequenced from 3'-end of transcripts (Fu et al., 2011). Trimmed tags were then mapped to the Arabidopsis genome (TAIR10) using Bowtie (0.12.7) (Langmead et al., 2009), allowing 2 mismatches. Only uniquely mapped tags were kept for downstream analyses. Uniquely located tags with six or more continuous adenines downstream from the poly(A) junction in a 10-nt window were identified as internal priming tags and discarded (Shepard et al., 2011).
Genome_build: TAIR10
Supplementary_files_format_and_content: wig files were generated using MACS program
|
| |
|
| Submission date |
Jan 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
xiaofeng cao |
| E-mail(s) |
xfcao@genetics.ac.cn
|
| 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 |
GPL13222 |
| Series (1) |
| GSE39051 |
Integrative genome-wide analysis reveals HLP1, a novel RNA-binding protein， regulates plant flowering by targeted alternative polyadenylation |
|
| Relations |
| SRA |
SRX220722 |
| BioSample |
SAMN01907320 |
