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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 12, 2019 |
| Title |
NPC_Day5_Rep1_4SUseq |
| Sample type |
SRA |
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| Source name |
4SU incorported RNA prepared from cells at day 5 of ES to NPC differentiation (parental mESCs - 46c Sox1-GFP reporter line)
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES-derived neural progenitor cells (differentiation day 5)
strain: 129/Ola
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| Growth protocol |
46C mouse embryonic stem cells (mESCs) derived from E14tg2A (129/Ola) containing a GFP insertion into the Sox1 locus (Ying et al., 2003; PMID: 12524553) were cultured on 0.1% gelatin (Sigma - catalogue no. G1890) coated Corning flasks in GMEM BHK-21 (Gibco - catalogue no. 21710-025) supplemented with 10 % foetal calf serum (FCS; Sigma - catalogue no. F-7524), 1,000 units/ml LIF, non-essential amino acids (Sigma - catalogue no. M7145), sodium pyruvate (Sigma - catalogue no. S8636), 50 μM 2-β-mercaptoethanol (Gibco - catalogue no. 31350-010) and L-glutamine. ESCs were differentiated into NPCs essentially as described previously (Ying et al., 2003 PMID: 12524553 and Pollard et al., 2006 PMID: 17141035). To ensure a homogeneous starting population of ESCs, cells were harvested using trypsin 24 h prior to initiating differentiation and seeded at 3x106 on 0.1% gelatin coated Corning flasks. Differentiation media (1:1 DMEM/F12:Neurobasal medium (Gibco – catalogue no. 31330-032 and 21103-049 respectively) supplimented with 0.5x B27 (Invitrogen – catalogue no. 17504044), 0.5x N2 (Invitrogen –catalogue no. 17502048), L-Glutamine and 50 μM 2-β-mercaptoethanol (Gibco - catalogue no. 31350-010)) was prepared fresh on the day of differentiation. On day 0, ESCs were harvested, washed twice with PBS and twice with differentiation media then seeded at a density of 1x106 ESCs on 0.1% gelatin coated T75 Corning flasks (gelatin coated at RT for 3-6 h and washed twice with PBS prior to seeding). Cells were then cultured for 7 days with media changes each day after day 2. NPC differetiation was monitored microscopically through the expression of the GFP reporter.
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| Extracted molecule |
total RNA |
| Extraction protocol |
4SUseq was performed essentially as described previously (Rabani et al., 2011 PMID: 21516085). Briefly, 4-thiouridine (4SU; Sigma - catalogue no. T4509) was added to ESCs / NPCs in culture to a final concentration of 500 µM and incubated at 37°C for 20 min. Cells were harvested by trypsinisation and washed twice with PBS at RT. Total RNA was isolated from 5x106 cells using trizol according to the manufacturer’s instructions (Invitrogen - catalogue no. 15596026). Following precipitation, purified RNA was resuspended in 100 μl RNase-free water and DNase treated using the TURBO DNA-free kit according to the manufacturer’s instructions (Invitrogen - catalogue no. AM1907M). Residual inactivation beads were removed by spinning the RNA sample through a QIAshredder column at 1000 g for 1 min (Qiagen- catalogue no. 79654). Following quantification, 50 μg of RNA was incubated for 1.5 h at RT with 100 μg of Biotin-HPDP (Pierce - catalogue no. 21341; reconstituted in Dimethylformamide at 1 mg/ml) in 1x Biotinylation Buffer (10 mM Tris pH 7.4 and 1 mM EDTA) to a total volume of 500 μl. Uncoupled biotin was removed through two consecutive rounds of 1:1 v/v chloroform extraction followed by isopropanol/NaCl precipitation. RNA was resuspended in 100 μl of RNase free water and mixed 1:1 w/w with µMacs Streptavidin beads (miltenyi - catalogue no. 130-074-101) and incubated for 15 min at RT with rotation. The RNA / bead mixture was applied to a µMacs column following pre-equilibration with wash buffer (100 mM Tris pH 7.5, 10 mM EDTA, 1 M NaCl and 0.1% Tween20). The captured beads were then washed with 3 x 900 µl of 65 oC wash buffer and 3 x 900 µl RT wash buffer. RNA was then eluted from the column by adding two consecutive rounds of 100 mM DTT. The eluate was added to 700 µl Buffer RLT (RNeasy MinElute Cleanup Kit; Qiagen catalogue no. 74204) and then purified according to the manufacturer’s instructions. Prior to library preparation, ribosomal RNA was depleted from the purified 4SU incorporate RNA using the low Input RiboMinus Eukaryote System v2 kit according to the manufacturer’s instructions (Ambion - catalogue no. A15027).
Libraries were constructed using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina according to the protocol for ribosome depleted RNA and with a 10 min RNA fragmentation step (NEB - catalogue no. E7760). Library PCRs were supplemented with 2x SYBR dye (Sigma – catalogue no. S9430) so that amplification could be monitored by quantitative PCR on a Roche lightcycler 480. To allow for sample multiplexing, PCRs were performed using index primers (NEBNext Multiplex Oligos for Illumina - Set 1. Catalogue no. E7335) and amplified to linear phase. Libraries were purified and then combined into 4 sample equimolar pools containing the indexes 1, 3, 6 and 8.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
4SU incorporated RNA
The library was barcoded with NEBNext® Multiplex Oligos for Illumina® - Index Primers Set 1 (Catalogue no. E7335S). Primer: INDEX_6. Barcode: GCCAAT.
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| Data processing |
Basecalling and quality scoring was performed directly using the NextSeq 550 software (RTAv2).
Base called data was de-multiplexed and converted in to FASTQ files using 'bcl2fastq' v2.20.0.422 with the following parameters: Allowed barcode mismatches - 0 Minimum trimmed read length - 35 Mask short adapter reads - 35 Adapter stringency - 0.9 Adapter trimming method - Sliding window Ignore missing BCLs - YES Ignore missing filters - YES Ignore missing positions - YES Ignore missing controls - YES Include non-PF clusters - NO Create FASTQs for index reads - NO
Alignment. For each de-multiplexed sample, multiple fastq files were merged using the ‘cat’ function for both read 1 and 2. Merged files were the aligned to the mouse genome (mm9) using bowtie2 v2.2.6 with standard parameters for paired end sequence data to generate .SAM files.
Further processing. Aligned read data was processed using HOMER v4.8. SAM files were converted into tag directories using ‘makeTagDirectory’ with the following parameters: -format sam -flip –sspe. Genomic intervals which extended beyond the end of the chromosomes were removed using ‘removeOutOfBoundsReads.pl’. Strand specific browser track files (bigwig format; ‘.bigWig’ or ‘.bw’) for each replicate were generated using ‘makeUCSCfile’ with the following parameters: -fsize 1e20 -strand + (or -) -norm 1e8 -color 25,50,200. Browser tracks of merged replicates were generated by combining replicate tag directories with the ‘makeTagDirectory’ function and then running ‘makeUCSCfile’ on the combined data as described above.
Genome_build: mm9
Supplementary_files_format_and_content: Processed files are in bigwig format. Those with extension '.bigWig' represent individual 4SU-seq replicates and those with ‘.bw‘ are combined replicates. Positive and negative strand files are denoted by ‘*PosStr*‘ or ‘*NegStr*‘ respectively.
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| Submission date |
Jun 13, 2018 |
| Last update date |
Jan 13, 2019 |
| Contact name |
Rob S Illingworth |
| E-mail(s) |
robert.illingworth@ed.ac.uk
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| Phone |
01316519640
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| Organization name |
The University of Edinburgh
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| Department |
Centre for regenerative Medicine
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| Lab |
Illingworth
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| Street address |
Centre for Regenerative Medicine
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| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH16 4UU |
| Country |
United Kingdom |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE89557 |
A new model for long-range chromatin reorganization linked to enhancer activation |
| GSE115774 |
Transcriptional profiling by 4SU-seq in mouse ESCs and ESC-derived neural progenitor cells. |
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| Relations |
| BioSample |
SAMN09407982 |
| SRA |
SRX4212629 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3189535_Day5_R1_NegStr.bigWig |
308.8 Mb |
(ftp)(http) |
BIGWIG |
| GSM3189535_Day5_R1_PosStr.bigWig |
315.6 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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