|
| Status |
Public on Feb 03, 2020 |
| Title |
U6 snRNA RiboMeth-Seq from B-LCL cell line originating from a boy affected by the Alazami syndrome |
| Sample type |
SRA |
| |
|
| Source name |
Blood lymphocytes
|
| Organism |
Homo sapiens |
| Characteristics |
origin: human blood
purification: U6 snRNA enriched by pulldown with biotinylated antisense oligonucleotides from total RNA
|
| Growth protocol |
B-LCLs cells were cultivated in RPMI-1640 with L-glutamine supplemented with 15% fetal bovine serum. Cells were grown under standard conditions at 37°C in 5% CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol reagent an the RNAs of interest were purified by two consecutive pulldowns with a mix of three biotinylated antisense oligonucleotides and dynabeads M-270 streptavidin (Invitrogen).
RNA samples purified by antisense oligonucleotide pulldowns were fragmented by alkaline hydrolysis in 50 mM Na2CO3 pH 9.0 for 50-55 minutes at 90°C. RNA fragments were resolved on polyacrylamide (acrylamid/bisacrylamid 19:1) urea gels, stained with SYBR gold and gel slices containing RNA fragments in the range between 21 nt and 35 nt were excised. Upon elution and precipitation of the fragments, the RNA was treated with 0.5 U/ml calf intestinal alkaline phosphatase (NEB) for 1 h at 37°C. Dephosphorylated RNAs were purified by phenol/chloroform/isoamyl alcohol extraction followed by precipitation. Phosphorylation of the 5’ ends occurred with the T4 polynucleotide kinase (Thermo Scientific), followed by heat-inactivation of the enzyme, removal of unincorporated ATPs via Illustra MicroSpin G-25 gel filtration columns, phenol/chloroform/isoamyl alcohol extraction of the RNA fragments and precipitation. Small RNA libraries were generated by ligating an adenylated adapter, [5’-App-TGGAATTCTCGGGTGCCAAGG-(C7-amino)-3’], to the 3’ end of the RNA fragments by a truncated T4 RNA Ligase 2. Ligated fragments were gel purified prior to ligation to the 5’ RNA adapter (5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’) using the T4 RNA Ligase 1 (NEB). After reverse-transcription with a specific primer, the cDNA was amplified by PCR, gel-fractionated and the region above empty adapter dimers was cut out. PCR products from these gel slices were eluted in elution buffer, precipitated and dissolved in water. The quality of the libraries was assessed by measurement on a TapeStation 4200 device.
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
B-LCL Alazami replicates 1-2
small RNA sequencing data
processed data file: RMS_Alazami_U6_B-LCL.xlsx
B-LCL_Alazami_rep1
|
| Data processing |
Base calling for HiSeq runs were performed using CASAVA 1.8.2 software
Base calling for MiSeq runs were performed using MiSeq Control Software 2.6.2.1
Adapter trimming was performed using cutadapt version 1.17 with parameters --overlap=8 --minimum-length=15 --discard-untrimmed
Mapping against U6 and U2 fasta sequences were performed using Bowtie 2 version 2.3.4.1
Further analysis were performed in R version 3.3.3 using the ShortRead 1.32.1 package according to Birkedal et. al. "Profiling of Ribose Methylations in RNA by High-Throughput Sequencing". Angew Chem Int Ed Engl. 2015 Jan 7;54(2):451-5
Genome_build: hg38
Supplementary_files_format_and_content: Excel files with RiboMeth-seq scores A and B
|
| |
|
| Submission date |
Dec 12, 2019 |
| Last update date |
Feb 03, 2020 |
| Contact name |
Gerhard Lehmann |
| E-mail(s) |
gerhard.lehmann@vkl.uni-regensburg.de
|
| Organization name |
University of Regensburg
|
| Department |
Biochemistry 1
|
| Street address |
Universitaetsstrasse 31
|
| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15520 |
| Series (2) |
| GSE126593 |
RiboMeth-Seq analysis of purified U6 and U2 snRNAs |
| GSE126911 |
The Alazami Syndrome-associated protein LARP7 guides U6 small nuclear RNA modification and contributes to splicing robustness |
|
| Relations |
| BioSample |
SAMN13548382 |
| SRA |
SRX7366952 |
