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| Status |
Public on Oct 30, 2017 |
| Title |
ControlTGIRTInputRep1 M1A IP |
| Sample type |
SRA |
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| Source name |
HEK293T cells
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| Organism |
Homo sapiens |
| Characteristics |
treatment: M1A IP
cell line: HEK293T
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| Treatment protocol |
For overexpression, plasmids encoding full length of TRMT6/TRMT61A under CMV promoter were obtained from ORIGENE. The plasmids were transfected into HEK293T cell using PolyJet (SignaGene) with one boost of the plasmid at 24 hours. Cells were harvested 48 hours following transfection.
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| Growth protocol |
Human HEK293T cells were plated in 6-well plates at 20% confluence.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Briefly, 40 μl of protein-G magnetic beads were washed and resuspended in 200 μl of IPP buffer, and tumbled with 5 μl of affinity purified anti-m1A polyclonal antibody (Synaptic Systems) at room temperature for 30 minutes. RNA was added to the antibody-bead mixture, and incubated for 2 h at 4°C. The RNA was then washed twice in 200 μl of IPP buffer, twice in low-salt IPP buffer (50 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5), and twice in high-salt IPP buffer (500 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5), and eluted in 30 μl RLT (Qiagen). To purify the RNA, 20 μl MyOne Silane Dynabeads (Life Technologies) were washed in 100 μl RLT, resuspended in 30 μl RLT, and added to the eluted RNA. 60 μl 100% ethanol was added to the mixture, the mixture attached to the magnet and the supernatant discarded. Following two washes in 100 μl of 70% ethanol, the RNA was eluted from the beads in 10 μl H20.
RNA was first subjected to FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), followed by a 3’ ligation of an RNA adapter using T4 ligase (New England Biolabs). Ligated RNA was reverse transcribed either using SuperScript-III (Invitrogen) or using TGIRT-III (InGex), and the cDNA was subjected to a 3’ ligation with a second adapter using T4 ligase. The single-stranded cDNA product was then amplified for 9-12 cycles in a PCR reaction. Libraries were sequenced on Illumina Nextseq platforms generating short paired end reads, ranging from 25-55 bp from each end.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Reads were aligned to the human genome (hg19) using STAR aligner with default parameters
An in house script was used to merge left and right reads into a single bam file spanning the full interval from the beginning of the 'left' read to the end of the 'right' read
Strand specific bedgraph files were generated quantifying the number of reads beginning at each position, using bedtools genomecov (activating the '-5' and '-strand' flags
Genome_build: hg19
Supplementary_files_format_and_content: bedgraph
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| Submission date |
Apr 05, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Schraga Schwartz |
| Organization name |
WEIZMANN INSTITUTE OF SCIENCE
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| Street address |
Herzl 234, Department of Molecular Genetics
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| City |
Rehovot |
| State/province |
Choose a State or Province |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE97419 |
Single-nucleotide mapping of N1-methyladenosine reveals a tissue-specific role in translational repression |
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| Relations |
| BioSample |
SAMN06688620 |
| SRA |
SRX2709995 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2564293_ControlTGIRTInputRep1_IP_minus.bed.gz |
20.0 Mb |
(ftp)(http) |
BED |
| GSM2564293_ControlTGIRTInputRep1_IP_plus.bed.gz |
20.4 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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