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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 25, 2011 |
| Title |
human_liver |
| Sample type |
SRA |
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| Source name |
liver
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| Organism |
Homo sapiens |
| Characteristics |
tissue: liver
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| Treatment protocol |
PolyA+ RNA was isolated using the Dynabead mRNA Purification Kit according to the manufacturer's instructions (Invitrogen, cat # 610.06)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
2 µL 0.1 uM tailed dT primer T(10)VN was combined with 150 ng polyA+ RNA in a final volume of 11 µL; for the human noVN experiment, the primer consisted only of T(10). The primer-template mix was heated at 65 °C for 5 min and chilled on ice before adding 9 µL reverse transcription master mix (4 µL 5x buffer, 2 µL 10 mM dNTPs, 1 µL 100 mM DTT, 1 µL RNaseOUT and 1 µL SuperScript III enzyme). The 20 µL reverse transcription reaction was incubated at 40 °C for 90 min, 70 °C for 15 min and cooled to 4 °C. RNA template was degraded by adding 1 µL RNase H (Invitrogen Corp.) and incubating at 37 °C for 20 min, 75 °C for 15 min and cooling to 4 °C. DNA was subsequently purified using the QIAquick PCR Purification kit and eluted with 65 µL elution buffer (Qiagen, Inc.). For second-strand synthesis, 60 µL purified cDNA was added to 40 µL Klenow master mix (12 µL water, 10 µL 10x NEBuffer 2, 5 µL 10 mM dNTPs, 3 µL 5 units/ul exo- Klenow fragment; M0212L,New England Biolabs, Inc.) and 10 µL 10 uM tailed random hexamer primer. The 100 µL reaction was incubated at 37 °C for 30 min and cooled to 4 °C. DNA was purified from the second-strand reaction by incubating with 1.8 volumes of Agencourt AMPure XP beads (Beckman Coulter) for 5 minutes, washing twice with 70% EtOH and eluting with 50 µL of elution buffer. This step greatly reduced the number of clones with inserts less than 40 nucleotides. For PCR amplification, 33 µL of purified second-strand synthesis reaction was combined with 17 µL of PCR master mix (10 µL 5x Buffer 2, 1 µL 25 mM MgCl2, 1 µL 10 mM dNTPs, 2 µL 10 uM forward primer, 2 µL 10 uM reverse primer, 1 µL ExpandPLUS enzyme; Roche Diagnostics Corp.). Samples were denatured for 2 min at 94 °C followed by 2 cycles of 94 °C for 10 s, 40 °C for 2 min, 72 °C for 1 min; 8 cycles of 94 °C for 10 s, 60 °C for 30 s, 72 °C for 1 min; 15 cycles of 94 °C for 15 s, 60 °C for 30 s, 72 °C for 1 min; and 72 °C for 5 min to polish ends before cooling to 4 °C. Double-stranded DNA was purified using AMPure XP beads as described above.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Alignments. Using Soap2, reads were aligned to the respective genome (human: hg19 if so indicated in the file name, hg18 otherwise; rhesus: rheMac2; mouse: mm9; rat: rn4; dog: canFam2) and to splice junctions from all available known (Refseq, UCSC Known Genes, Ensembl, ESTs) and predicted transcripts (Genscan, N-Scan), and to junctions formed from one- and two-exon-skipping events predicted in these transcripts. Only reads aligning to unique loci were retained.
Filtering. A modified PolyA-Seq assay was performed on a UHR sample, using a T(10) primer ("noVN") instead of T(10)VN. Genomic alignments was performed with Blat. Reads with perfectly aligned 3' ends (except those matching ends of mRNAs or ESTs, or known polyA sites) were considered to locate internal priming sites, while reads with unalignable 3' As were deemed to identify true polyA sites. A frequency matrix for the 10 bases downstream of all alignments was created for each of these two sets of sites. These matrices were used to score the 10-mer downstream of every read alignment from previous samples. Alignments were retained if the log of the odds (true polyA site / internal priming site) was 3 or greater.
Peak/site detection. Following filtering, ends of reads within 25 bp of each other on the same strand were clustered.
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| Submission date |
Jun 24, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Adnan Derti |
| E-mail(s) |
adnan.derti@gmail.com
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| Organization name |
personal
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| Department |
Bioinformatics
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| Street address |
100 Alden Street
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| City |
Dedham |
| State/province |
MA |
| ZIP/Postal code |
02026 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE30198 |
Digital gene expression and global mapping of polyadenylation sites with PolyA-Seq |
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| Relations |
| SRA |
SRX080221 |
| BioSample |
SAMN00631260 |
| Named Annotation |
GSM747472_human_liver.alignments.sum.bed.gz |
| Named Annotation |
GSM747472_human_liver.alignments.sum.hg19.bed.gz |
| Named Annotation |
GSM747472_human_liver.sites.clustered.bed.gz |
| Named Annotation |
GSM747472_human_liver.sites.clustered.hg19.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM747472_human_liver.alignments.sum.bed.gz |
8.2 Mb |
(ftp)(http) |
BED |
| GSM747472_human_liver.alignments.sum.hg19.bed.gz |
8.0 Mb |
(ftp)(http) |
BED |
| GSM747472_human_liver.sites.clustered.bed.gz |
1.0 Mb |
(ftp)(http) |
BED |
| GSM747472_human_liver.sites.clustered.hg19.bed.gz |
980.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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