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Status |
Public on Sep 13, 2016 |
Title |
2OMe-seq 1mM dNTPs (HeLa, Method #2) |
Sample type |
SRA |
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Source name |
HeLa S3
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Organism |
Homo sapiens |
Characteristics |
cell line: HeLa S3 cell type: cervical adenocarcinoma
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Biomaterial provider |
ATCC (cat. CCL2.2)
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Treatment protocol |
none
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Growth protocol |
HeLa S3 cells were obtained from ATCC (cat. CCL2.2), and cultured in DMEM (4.5g/L D-Glucose), supplemented with 10% FBS, 0.1mM NEAA, 25U/ml penicillin, and 25 μg/ml streptomycin.
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Extracted molecule |
total RNA |
Extraction protocol |
Cells were washed twice in 1X PBS, and lysed by addition of 1ml ice-cold TRIzol (Invitrogen). RNA integrity measurements were performed using Fragment Analyzer™ (Advanced Analytical). All samples had RNA Quality Number (RQN) greater than 9.8. Briefly, 2 μg of total RNA were fragmented by addition of 5X First Strand Buffer (Invitrogen), and incubation at 95°C for 5 minutes. Fragmented RNA was purified on RNA Clean & Concentrator™-5 columns (Zymo Research). The eluted RNA was end repaired by adding 20U of T4 PNK (NEB), and incubating at 37°C for 1h, and then purified again on RNA Clean & Concentrator™-5 columns. A pre-adenylated (rApp) adapter (AGATCGGAAGAGCACACGTCT) was ligated to the end-repaired RNA fragments using 400U of T4 RNA Ligase 2, Deletion Mutant (Epicentre) in the presence of 20% PEG-8000, by incubating at 16°C for 18 hours. The day after, reactions were purified on RNeasy Micro Spin columns (Qiagen) to get rid of the excess adapter. Following cleanup, adapter-ligated RNA fragments were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 200-250nt fragments was cut. RNA was recovered by passive diffusion in RNA Diffusion Buffer [450mM Ammonium Acetate, 0.05% SDS] for 16 hours at 4°C with end-to-end rotation. Following isopropanol precipitation, reverse transcription was performed in a 20 μl reaction, using 1mM final dNTPs (High dNTP sample), 0.5U/μl AMV Reverse Transcriptase (NEB), and an oligonucleotide complementary to the 3’ adapter (AGACGTGTGCTCTTCCGATCT). The reverse transcription reaction was conducted in 30 minutes at 42°C, followed by 10 minutes at 95°C to inactivate the AMV enzyme. Template RNA was degraded by addition of 1 μl of Ribonuclease H (Ambion), and 1 μl of RNase Cocktail Enzyme Mix (Ambion), followed by 30 minutes incubation at 37°C. cDNAs were purified on RNA Clean & Concentrator™-5 columns (Zymo Research), separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 50-150nt fragments (corresponding to truncated reverse transcription products) was cut. DNA was recovered by passive diffusion in TE Buffer [10mM Tris-HCl, 1mM EDTA] for 16 hours at 37°C with moderate shaking. Following ethanol precipitation, an adapter corresponding to the reverse complement of the standard Illumina TruSeq Small RNA 5’ Adapter (GATCGTCGGACTGTAGAACTCTGAAC), modified with a 5’-P group and a 3’-C3 spacer, was ligated to cDNAs 3’-OH termini using 200 U of CircLigase II for 6 hours at 65°C. The adapter-ligated cDNAs were then subjected to 18 cycles of PCR using standard Illumina TruSeq primers, and excess primers were removed using Agencourt Ampure XP beads. Libraries were pooled in equimolar amounts, and subjected to sequencing on the Illumina™ NextSeq 500 Sequencer.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
Library strategy: 2OMe-seq Raw reads were converted to FastQ, and examined using the FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). For each run, the first 5 nucleotides at 5’-end were trimmed using the fastx_trimmer tool of the FASTX-Toolkit suite. Reads were then clipped from 3’ adapter sequences (requiring a minimum match of 10 nucleotides), using the fastx_clipper tool of the FASTX-Toolkit suite, discarding reads shorter than 25 nucleotides (parameters: -a TGGAATTCTCGGGTGCCAAGG -l 25 -M 10 -Q 33; -a AGATCGGAAGAGCACACGTCT was replaced for Alkaline hydrolysis and 2OMe-seq Method #2 libraries, due to the different 3’ adapter used). Reads were mapped to the reference transcriptome, composed of 28S and 18S rRNA sequences, plus sequences of the 3 in vitro generated spike-ins (where included), using Bowtie v1.1.1 (parameters: -q -n 2 --norc -m 1 --best --strata). Genome_build: Reads were mapped only to 18S and 28S rRNA sequence for each species. Accessions are as follows: Homo sapiens 18S (NR_003286), Homo sapiens 28S (NR_003287), Mus musculus 18S (NR_003278), Mus musculus 28S (NR_003279). Supplementary_files_format_and_content: Processed data file in BEDGraph format are provided (Columns: Transcript ID, Position, Position +1, Number of reads mapping 1nt downstream).
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Submission date |
Sep 15, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Danny Incarnato |
E-mail(s) |
d.incarnato@rug.nl
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Organization name |
University of Groningen
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Department |
Molecular Genetics
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Street address |
Nijenborgh 7
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City |
Groningen |
State/province |
Netherlands |
ZIP/Postal code |
9747 AG |
Country |
Netherlands |
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Platform ID |
GPL18573 |
Series (1) |
GSE73065 |
High-throughput single-base resolution mapping of 2’-O-Methylated residues |
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Relations |
BioSample |
SAMN04088187 |
SRA |
SRX1247367 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1881615_Library5.bedgraph.gz |
38.3 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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