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| Status |
Public on Jul 10, 2017 |
| Title |
TERRA capture RNA-Seq_TeloPrimer |
| Sample type |
SRA |
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| Source name |
TERRA capture RNA-Seq
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| Organism |
Mus musculus |
| Characteristics |
strain: CAST/Ei x 129/Sv/Jae
cell type: Embryonic stem cells
primers: Telomeric Primer
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| Treatment protocol |
n/a
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| Growth protocol |
Cells were cultured in regular ES medium (15% FBS) with LIF
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by Trizol followed by acid phenol/chloroform extraction. 20 mg of total RNA was treated with 4 units of TURBO DNase at 37°C for 10 min in 100 ml of RNase inhibitor (10 nM of Ribonucleoside Vanadyl Complex(VRC) ) containing buffer. EDTA (final concentration 5 mM) was added to disrupt VRC, and DNase-treated RNA was purified by Trizol. 10 pmol of biotin-labeled oligo probes ((TAACCC)3TA-/3’BioTEG) was added to 20 mg of RNA, and the RNA-probes mixture was denatured at 70°C in 100 ml of 6XSSC hybridization buffer for 10 min. Denatured RNA was directly transferred to 44°C heat block and incubated for 30 min. RNA was then captured by 100 ml of MyOneC1 beads at 37°C for 15 min. RNA-beads were washed with 2XSSC/0.1% NP40 at 37°C for 4 times (5 min each time), 1XSSC/0.1 NP40 at 37°C twice, and then RNA-beads were rinsed once with 1X SSC at room temperature. TERRA-enriched RNA was eluted in 30 ml of DEPC-treated water at 70°C for 5 min.
cDNA libraries were reversed transcribed with telomeric repeat primers (TAACCC)4 using Superscript III (Invitrogen) at 55°C for 60 min. 1.25 mM of dUTP was supplied during second strand synthesis, and then dsDNA was briefly sonicated by covaris E220 in microTUBEs (Duty factor=2%, peak intensity power=140, cycle per burst=200, time=15 second). End repair, dA-tailing, adaptor ligation, and USER enzyme digestion were performed according to NEBUltra Directional RNA library preparation protocol for Illumina (NEB). Larger fragments (~>500 bp) of adaptor-ligated DNA were selected by 0.6x AMpure XP beads. Sequencing of purified libraries was carried out on an Illumina MiSeq instrument for paired 300 nucleotides reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
TERRA RNA
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| Data processing |
mm10
Basecalls performed using Illumina MiSeq software. Reads aligned to mouse reference genomes (GRCm38/mm10) using Tophat2 after removal of adaptor sequences by Trim Galore. Due to the low quality after 100 cycles of Miseq runs, it produced less mapped reads using longer reads (300-paired) than shorter reads (100-paired). We presented here using the first 100-paired end reads alignment instead of 300-paired end alignment. After removal of PCR duplicates, unmapped reads were filtered out. Alignments were divided into positive and negative strands, followed by coverage generation. Scaled wig files were generated by dividing these coverages by number of fragments per million (FPM).
Alignments were divided into positive and negative strands, followed by coverage generation. Scaled bigwig files were generated by dividing these coverages by number of fragments per million (FPM).
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| Submission date |
Dec 08, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Hsueh-Ping Chu |
| Organization name |
MGH, Harvard Medical School
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| Department |
molecular biology
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| Lab |
Jeannie T. Lee
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| Street address |
185 Cambridge St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (1) |
| GSE69887 |
PAR-TERRA RNA directs homologous sex chromosome pairing |
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| Relations |
| SRA |
SRX1471400 |
| BioSample |
SAMN04326934 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1968164_TERRASeq_TelPrimer.negStrand_fpm.bw |
1.7 Mb |
(ftp)(http) |
BW |
| GSM1968164_TERRASeq_TelPrimer.posStrand_fpm.bw |
1.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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