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| Status |
Public on Sep 10, 2018 |
| Title |
Genome-wide target profiling of pluripotency-associated long noncoding RNAs by RAT-Seq |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
Using RNA reverse transcription-associated trap sequencing (RAT-Seq), we profiled the genome-wide binding gene targets for pluripotency-associated long noncoding RNAs (lncRNAs), including Platr10 (Osclr8), Oeblr20, Oplr16, Palr35, Palr34, Peln1, and Peln4. These datasets will help study the mechanisms underlying the role of lncRNAs in the establishment and maintenance of pluripotency.
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| Overall design |
A RNA reverse transcription-associated trap sequencing (RAT-seq) assay [1, 2] was modified to map the genome-wide interacting gene targets for lncRNAs that are differentially expressed in reprogramming of fibroblasts into pluripotency. Cells were collected at different stages of pluripotent reprogramming, including fibroblasts and induced pluripotent stem cells (iPSCs) [3, 4]. After cross-linking with 2% formaldehyde to fix the lncRNA-chromatin structure, cells were lysed with cell lysis buffer (10 mM Tris [pH 8.0], 10 mM NaCl, 0.2% NP-40, 1x protease inhibitors). After centrifugation, nuclei were suspended in 1× reverse transcription buffer in the presence of 0.3% sodium dodecyl sulfate (SDS), and incubated at 37°C for 1 hr. Triton X-100 was then added to a final concentration of 1.8% to sequester the SDS.
The gene strand-specific reverse transcription was performed using lncRNA-specific complementary primers in the presence of biotin-dCTP. After 50 min of reverse transcription of Platr10 lncRNA with Maxima Reverse Transcriptase (Thermo Fisher Scientific, CA) at 62 C, the reaction was stopped by adding 4µl 0.5M EDTA. After nuclear lysis, the chromatin complex was subjected to sonication for 180 s (10 s on and 10 s off) on ice with a Branson sonicator with a 2-mm microtip at 40% output control and 90% duty cycle settings. The biotin-labeled cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA). After reversing the cross-links and washing with 10 mg/ml proteinase K at 65°C overnight and treatment with 0.4 µg/ml RNase A for 30 min at 37°C, the genomic DNA that interacts with the lncRNA was extracted and digested by Dpn I, and ligated with the NEBNext adaptors (NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina) to construct the library. The library DNAs were subject to Illumina sequencing (Shanghai Biotechnology, Shanghai).
As the controls, the RAT assay was performed for iPSCs and fibroblasts by replacing lncRNA-specific complimentary primers with random primers and constructed a control library for sequencing using the same protocol.
To reduce the background, the RAT-Seq data were normalized over the peaks of the control RAT-Seq data that were generated by using random oligonucleotide primers in the RAT assay. For this, differential binding analysis was performed with the DiffBind package using parameters of fold change difference ≥2 and p-value < 0.05, with false discovery rate (FDR) <0.1. The adjusted RAT-Seq data were used for mapping the lncRNA target gene interaction network.
LncRNAs and controls:
1. Platr10 (ENSMUSG00000099370, NONMMUT043505, Osclr8, Oct4-Sox2 coating lncRNA 8): a pluripotency-associated lncRNA that is activated in pluripotent reprogramming. It binds to the regulatory elements of multiple stem cell core factor genes.
2. Palr35 (ENSMUSG00000103968): a lncRNA that is activated in pluripotent reprogramming. However, it does not interact with the promoters of stem cell core factor genes.
3. Oeblr20 (NONMMUG017962, Oct4 enhancer binding lncRNA 20): a pluripotent lncRNA that is activated in pluripotent reprogramming. It binds to the enhancer elements of stem cell core factor gene Oct4.
4. Oplr16 (NONMMUT101010): a pluripotent lncRNA that is activated in pluripotent reprogramming. It binds to the promoter of stem cell core factor gene Oct4.
5. Peln4 (NONMMUG024191): a pluripotency exit-associated lncRNA that is expressed in somatic cells, like fibroblasts. It becomes silenced in pluripotent reprogramming.
6. Peln1 (NONMMUG035018): a pluripotency exit-associated lncRNA that is expressed in somatic cells, like fibroblasts. It becomes silenced in pluripotent reprogramming.
7. RAT-seq random control in iPSC: A RAT-seq control library that is constructed using random oligonucleotide primers in iPSCs.
8. RAT-seq random control in FIB: A RAT-seq control library that is constructed using random oligonucleotide primers in fibroblasts.
References:
1. Sun J, Li W, Sun Y, Yu D, Wen X, Wang H, Cui J, Wang G, Hoffman AR, Hu JF: A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies. Nucleic Acids Res 2014, 42:9588-9601.
2. Wang H, Li W, Guo R, Sun J, Cui J, Wang G, Hoffman AR, Hu JF: An intragenic long noncoding RNA interacts epigenetically with the RUNX1 promoter and enhancer chromatin DNA in hematopoietic malignancies. Int J Cancer 2014, 135:2783-2794.
3. Zhang H, Jiao W, Sun L, Fan J, Chen M, Wang H, Xu X, Shen A, Li T, Niu B, et al: Intrachromosomal looping is required for activation of endogenous pluripotency genes during reprogramming. Cell Stem Cell 2013, 13:30-35.
4. Du Z, Jia L, Wang Y, Wang C, Wen X, Chen J, Zhu Y, Yu D, Zhou L, Chen N, et al: Combined RNA-seq and RAT-seq mapping of long noncoding RNAs in pluripotent reprogramming. Sci Data 2018, 5:180255.
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| Contributor(s) |
Du Z, Zhu Y, Celik I, Chen J, Wang Y, Jia L, Wang C, Li W, Hoffman AR, Cui J, Hu J |
| Citation(s) |
23747202, 30457566, 31903125, 32055844, 34412677 |
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| Submission date |
Jul 21, 2017 |
| Last update date |
Aug 24, 2021 |
| Contact name |
Jifan Hu |
| E-mail(s) |
jifan@stanford.edu
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| Phone |
6508523175
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| Organization name |
Stanford University Medical School
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| Department |
Palo Alto Veterans Institute for Research
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| Lab |
Bldg 100, Room E4-200
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| Street address |
3801 Miranda Ave, Bldg 100, Room E4-200
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| City |
Palo Alto |
| State/province |
CA |
| ZIP/Postal code |
94304 |
| Country |
USA |
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| Platforms (1) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA395379 |
| SRA |
SRP113330 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE101765_RAW.tar |
263.6 Mb |
(http)(custom) |
TAR (of BEDGRAPH) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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