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| Status |
Public on Dec 18, 2013 |
| Title |
child patient T3 (microcephaly), mutation CLP1 R140H |
| Sample type |
SRA |
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| Source name |
mutation CLP1 R140H
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| Organism |
Homo sapiens |
| Characteristics |
cell type: primary fibroblasts
patient description: child patient T3 (microcephaly)
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| Treatment protocol |
none
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| Growth protocol |
Human fibroblasts were cultured at 37°C, 95% humidity and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 3 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin sulfate (all reagents from Sigma). Cells were split and/or harvested using 0.05% Trypsin–EDTA.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA for sequencing was isolated from patient (BAB3401 and 3402) or parental (BAB3845 and 3846) fibroblasts using the TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions
For each sample 300 ng of total RNA were hydrolyzed in a 15 μL buffer of 10 mM Na2CO3 and 10 mM NaHCO3 at ~pH 9.7, for 5 minutes at 90 °C. The hydrolyzed RNA was dephosphorylated with 10 U of calf intestinal phosphatase (NEB) in a 50 μL reaction of 100 mM NaCl, 50 mM Tris-HCl pH 7.9 at 25 °C, 10 mM MgCl2, 1 mM DTT, 3 mM Na2CO3 and 3 mM NaHCO3, at 37 °C for 1 h. The resulting RNA was re-phosphorylated with 10 U of T4 polynucleotide kinase (NEB) in a 20 µl reaction of 70 mM Tris-HCl pH 7.6 at 25 °C, 10 mM MgCl2, 5 mM DTT and 1 mM ATP, at 37 °C for 1 h. Fragments of 19-24 nts were converted into barcoded small RNA cDNA libraries and submitted for Illumina sequencing
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
barcoded small RNA-seq
BAB3402
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| Data processing |
Samples are demultiplexed by assigning by 5 nt barcodes and adapters and barcodes are removed from fastq files converted to fasta using perl scripts.
Fasta file is collapsed into a non-redundant fasta file in which the read count is contained within the read header and mapped against an in-house tRNA reference database derived from GtRNAdb, http://gtrna.ucsc.edu. BWA v0.5.9 used, with parameters aln -n 2 -t 16 -i 0 -d 0 to align reads to the reference.
Multi-mapping reads are first assigned to hits with the lowest number of errors, then the count is split equally between the number of locations mapped.
Genome_build: hg19
Supplementary_files_format_and_content: tab separated file summarizing reads attributed to an internal reference of tRNAs. First column is the read, second is the count, last column a comma-separated list of mapped annotation
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| Submission date |
Dec 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Miguel Brown |
| E-mail(s) |
miguel.a.brown@gmail.com
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| Organization name |
The Rockefeller University
|
| Street address |
1230 York Ave Box 186
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE53391 |
Human CLP1 mutations alter tRNA biogenesis affecting both peripheral and central nervous system function |
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| Relations |
| BioSample |
SAMN02462926 |
| SRA |
SRX393166 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1290725_BAB3402_geo.txt.gz |
1.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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