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Status |
Public on Mar 03, 2011 |
Title |
brain_RNAseq_Tdp43-oligo_rep2.1 |
Sample type |
SRA |
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Source name |
Striatum
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 age: 8-10 weeks treatment: Tdp-43 anti-sense oligo
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Treatment protocol |
8-10 week old female C57Bl/6 mice were anesthetized with 2.5-3% isofluorane, and a midline incision of approximately 1cm was made on their heads. Using stereotaxic guides, 3 mL of antisense oligonucleotide (ASO) solution targeting Tdp-43 or a control – corresponding to a total of 75 mg or 100 mg ASOs – or saline buffer was injected using a Hamilton syringe directly into their striatum. The incision was closed with sutures and the mice were allowed to recover in their cages. They were monitored for any adverse effects for the next two weeks until they were sacrificed. The striatum and adjacent cortex area were dissected, placed in separate tubes containing 1 mL Trizol (Invitrogen) and were frozen at -80oC until use.
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Extracted molecule |
total RNA |
Extraction protocol |
The RNA-seq libraries were constructed as described previously (Parkhomchuk, 2009) with slight modifications. Briefly, 7mg of total RNA was used for polyadenylated RNA selection using oligo-dT paramagnetic beads (Invitrogen). First-strand synthesis was prepared using a combination of oligodT primers and random hexamers with SuperScript III reverse transcriptase (Invitrogen), using gradually increasing temperature. dNTPs were then removed from the reaction by gel filtration through Sephadex G-50 (GE-health care) spin columns equilibrated with 1mM TrisHCl, pH 7.0. Second strand was then synthesized by a 2hr-incubation for 16oC with a dUTP-containing nucleotide mixture, 100mM MgCl2, 0.1M DTT, and a combination of ligase (NEB), DNA polymerase I (NEB) and RNase H (Invitrogen) in second strand synthesis buffer (Invitrogen). Double-stranded cDNA was then purified through spin columns (QIAGEN) and fragmented by ultrasonic digestion (Bioruptor, Diagenode) with continuous cooling at 1oC, using repeated cycles of 30 sec sonication with 100% amplitude and 30 sec breaks for 2-3 hrs. 1-2 mL of the material was used to check the level of fragmentation on a FlashGel DNA Cassette 1.2% (LONZA). End repair, A-tailing and adaptor ligation were all performed according to the Illumina protocol and libraries were subjected to electrophoresis on a 2% Low Range Ultra Agarose gel (BioRad). Fragments with a size range of 150-200 bp were cut out of the gel and libraries were gel purified on spin columns (QIAGEN) according to the manufacturer’s instructions. Prior to amplification, libraries were incubated with Uracil N-Glycosylase (Applied Biosystems) to digest the dUTP-containing strand, thereby ensuring a single orientation of our RNA-seq libraries. 8 pM of amplified libraries was used for sequencing on the Illumina GA2 for 72 cycles.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina Genome Analyzer II |
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Description |
strand-specific RNAseq
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Data processing |
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
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Submission date |
Feb 10, 2011 |
Last update date |
May 15, 2019 |
Contact name |
Gene Yeo |
E-mail(s) |
geneyeo@ucsd.edu
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Organization name |
UCSD
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Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
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City |
La Jolla |
State/province |
CA |
ZIP/Postal code |
92037 |
Country |
USA |
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Platform ID |
GPL9250 |
Series (2) |
GSE27218 |
Disrupted processing of long pre-mRNAs and widespread RNA missplicing are components of neuronal vulnerability from loss of nuclear TDP-43 (RNA-seq) |
GSE27394 |
Disrupted processing of long pre-mRNAs and widespread RNA missplicing are components of neuronal vulnerability from loss of nuclear TDP-43 |
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Relations |
SRA |
SRX043757 |
BioSample |
SAMN00216120 |
Supplementary file |
Size |
Download |
File type/resource |
GSM672541_MP15_2_brainTDP43KD19.mm8.bowtie.gz |
397.5 Mb |
(ftp)(http) |
BOWTIE |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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