|
| Status |
Public on Jul 25, 2016 |
| Title |
Ribosome protected fragments (RPF) of TSC2+/+ cells (number 16) treated with Rapamycin, Biological Replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
TSC2+/+ cells (number 16) treated with Rapamycin, RPF
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Embryonic stem cell-derived neurons
genotype/variation: TSC2+/+
treatment: Rapamycin
cell line id: 16
assayed molecule: Ribosome protected fragments
|
| Treatment protocol |
Cells were treated with DMSO, AZD-8055, and Rapamycin for 3 hours, respectively. After 3 hours cells are washed and harvested.
|
| Growth protocol |
NSCs were cultured according to standard methods. All used tissue culture dishes were coated with poly-L-ornithine (Sigma Aldrich) and laminin (Roche) and undifferentiated cultures were maintained in a basic medium composed of a 1:1 mix of DMEM:F12 Glutamax medium and Neurobasal medium (both Gibco, Invitrogen) that was supplemented with 1x B27, 1x N2 and 0.1 mM beta-mercaptoethanol (all Gibco, Invitrogen). For self-renewing conditions the following growth factors were added: 10 ng/mL FGF2, 20 ng/mL BDNF (both Peprotech) and 10 ng/mL EGF (R&D Systems). Ventralization was induced for a period of seven days by replating the cells at a density of 12000 cells/cm2 and changing the supplementing growth factors to 200 ng/mL Shh, 100 ng/mL FGF8 (both Peprotech) and 100 μM ascorbic acid phosphate (Sigma Aldrich). Neuronal differentiation was initiated by replating the cells at a density of 40000 cells/cm2 in basic medium supplemented with 20 ng/mL BDNF, 10 ng/mL GDNF (both Peprotech), 0.5 mM cAMP (BIOLOG Life Science) and 100 μM ascorbic acid phosphate (Sigma Aldrich). Medium was changed twice per week until the day of analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells for each biological replicate of heterozygous and homozygous cells with or without drug treatment were washed with ice cold PBS and lysed on ice in the presence of 100 μg/mL cycloheximide (Sigma). The cleared lysate was flash frozen and stored at -80° C until further processing. For ribosomal RNA depletion the RiboZero magnetic Gold kit (Illumina) was used. Quality of amplified libraries was accessed by capillary electrophoresis with a high sensitivity DNA chip on a 2100 Bioanalyzer (Agilent Technologies) and quantified by quantitative PCR with a sequencing library quantification kit (KAPA Biosystems) on a Roche Light Cycler 480. Multiplexed libraries with 1 % spiked in PhiX control were sequenced on a HiSeq2500 instrument for 50 cycles using version 4 chemistry reagents (Illumina).
Ribosome profiling and RNA sequencing libraries were prepared using the TruSeq Ribo Profile kit (Illumina, #RPHMR12126) as detailed in the manufacturer’s protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
16Rapamycin_RPF_1
16Rapamycin_L002
TSC2-treatment_RPF.txt
|
| Data processing |
Linker tags were removed from RNA sequencing and ribosome profiling reads by the FASTX Toolkit, v0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit/)
All reads that mapped to rRNAs, tRNAs or mitochondrial rRNAs were removed, and the remaining reads were mapped to RefSeq (v38) by TopHat v2.0.13.
Finally all read counts that mapped uniquely to genes were extracted for expression analysis with the help of samtools, v1.1.
Genome_build: GRCh37.p13
Supplementary_files_format_and_content: .txt files report raw read counts that mapped uniquely to genes
|
| |
|
| Submission date |
Mar 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jitao David Zhang |
| E-mail(s) |
jitao_david.zhang@roche.com
|
| Phone |
+41616886251
|
| Organization name |
F. Hoffmann-La Roche
|
| Department |
Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel
|
| Lab |
Pharmaceutical Sciences
|
| Street address |
Grenzacherstrasse 124
|
| City |
Basel |
| ZIP/Postal code |
4070 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE78960 |
Modeling the Neuropathology of Tuberous Sclerosis with Human Stem Cells Reveals a Role for Inflammation and Angiogenic Growth Factors [Treatment] |
| GSE78961 |
Modeling the Neuropathology of Tuberous Sclerosis with Human Stem Cells Reveals a Role for Inflammation and Angiogenic Growth Factors |
|
| Relations |
| BioSample |
SAMN04536318 |
| SRA |
SRX1617584 |
