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| Status |
Public on Jun 17, 2024 |
| Title |
TCPOBOP_1d_Male_STARRTYC3/7_RNA_replicate1 |
| Sample type |
SRA |
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| Source name |
liver
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| Organism |
Mus musculus |
| Characteristics |
tissue: liver
strain: Crl:CD1(ICR)
age at_euthanasia: 7-8 weeks
Sex: Male
treatment: TCPOBOP (3mg/kg), 1d
dosing regimen: Low Corn Oil
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| Treatment protocol |
In vivo transfection: Plasmid DNA to be delivered to each mouse was resuspended in TransIT-EE delivery solution (MIR 5340, Mirus, Inc, Madison, WI) in a final volume of ~3 ml, equivalent to 10% of the mouse body weight (vol/weight). Each mouse was injected with a volume corresponding to ~10% of the body weight, up a maximum of 3 ml (for mice > 30 g weight) and delivered within 5-8 seconds without appreciable resistance. Hydrodynamic injection was largely ineffective, as judged by Renilla luciferase activity assayed in liver extracts, when resistance to injection precluded delivery of the full volume or when the total time of injection exceeded 10 seconds. For STARR-seq studies, 15 ug of STARR-seq library plasmid DNA was mixed with 3 ug of Renilla reporter plasmid DNA was injected per mouse. TCPOBOP (1,4-bis(2-(3,5-dichloropyridyloxy))benzene) (Chem Cruze, cat. #SC-203291) was delivered to mice at 6 days after HDI using Low Corn Oil regimen. Low Corn Oil regimen: TCPOBOP was dissolved in DMSO at 7.5 mg/ml, and then diluted 10-fold into corn oil, followed by IP injection at 4 µl per g body weight (final dose: 3 mg TCPOBOP and 4 µl of 10% DMSO in corn oil, per kg body weight). Mice were euthanized 1 day after TCPOBOP or corn oil vehicle treatment for the groups. TCPOBOP or Vehicle injections were performed in the morning, and mice were euthanized between 10:30 AM and 12 noon under CO2 followed by cervical dislocation (Boston University animal facility light cycle: 7:30 AM to 7:30 PM). Livers were excised and flash frozen in liquid nitrogen then stored at -80° C.
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| Growth protocol |
Male and female CD1 mice (ICR strain), approx. 7 weeks old, were purchased from Charles River Laboratories (Wilmington, MA) and kept on a 12-hour light/dark cycle with food and water without restriction.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The extraction methods, adapted from the published STARR-seq protocol (Neumayr, Pagani et al. 2019), were used in a nested PCR amplification protocol to characterize the transcriptional activity of each DHS sequence (putative regulatory region) to be assayed by HDI-STARR-seq: 1) a library prepared from DNA extracted from flash frozen liver tissue collected 7 d after in vivo transfection by HDI (primers ON8381, ON8382); and 2) a library prepared from the transcribed RNA (reporter sequences) extracted from each HDI-transfected liver (primers ON8379, ON8380; see STARR_seq_primer.txt). STARR-seq reporter plasmid DNA remaining in the liver 7 d after HDI was extracted using a commercial QIAprep spin miniprep kit (QIAgen, cat. # 27106). Liver tissue (200 mg frozen liver) was initially homogenized in 750 ul of QIAgen Buffer P1 using a rubber-tipped micropestle and passed through a 20G needle 3 times to homogenize large tissue pieces. The extracted plasmid DNA was bound to a QIAprep spin column, eluted in EB buffer and used as a PCR template in a subsequent nested PCR reaction using primers ON8381 and ON8382 (see STARR_seq_primer.txt). To extract transcribed reporter RNA, 120 ug of total liver RNA was isolated from 60-100 mg liver tissue from each mouse by TRIzol guanidinium thiocyanate-phenol-chloroform extraction then purified by polyA selection using oligo(dT) beads (New England Biolabs, cat. #E7490L). The transcribed RNA was digested with Turbo DNase I (Ambion, cat. # AM2238) at 37°C for 30 min, reverse transcribed using primer ON8731 (see STARR_seq_primer.txt) for STARR-seq RNA first strand synthesis and Superscript III first-strand synthesis supermix (Invitrogen, cat. # 18080-400) and treated with 10 mg RNase A per ml (Fermentas, cat. # EN0531) for 1 h at 37 Celsius. The cDNA obtained was purified using 1.8x SPRI beads then used as a PCR template in the nested PCR reaction.
The nested PCR reaction requires a set of primers that specifically targets DNA or RNA derived from a STARR-seq reporter, after which the sequences are amplified using Illumina dual index primers. A primer set, primers ON8381 and ON8382 (see STARR_seq_primer.txt) was designed to amplify the STARR-seq plasmid DNA libraries by specifically targeting the unspliced intron incorporated into the original plasmid and present in the plasmid and DNA libraries, but that is spliced out of the reporter RNA sequences. Another primer set, primers ON8379 and ON8380 (see STARR_seq_primer.txt) straddles the splice junction and was designed to specifically amplify only the transcribed and spliced RNA molecules. Subsequently, nested PCR was completed using NEBNext® Multiplex Oligos for Illumina (#E7600S) for all three library types (plasmid, DNA and RNA) to further amplify the material and attach Illumina i5 and i7 barcoded sequences used for multiplex sequencing on an Illumina sequencer.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
polyA-selected RNA, stranded library
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| Data processing |
READ MAPPING: reads were aligned to the mm9 mouse genome using Bowtie2 (Langmead et al 2009, Genome Biology) with default settings. Reads were filtered so that only uniquely-aligned reads were used for downstream analysis.
PEAK CALLING: A set of 1,673 merged MACS2 peak regions were identified by MACS2 (Zhang et al 2008, Genome Biology) analysis of the associated plasmid, DNA, RNA libraries with default parameters without filtering for PCR duplicates. Peaks were filtered to remove ENCODE blacklisted regions (www.sites.google.com/site/anshulkundaje/projects/blacklists) and also regions called as peaks that contain only PCR duplicated reads.
READ COUNTING: Reads within the set of 1,673 MACS2 peak regions were counted using bedtools coverage option without filtering for PCR duplicates.
Assembly: mm9
Supplementary files format and content: G187_focus_processed_dataset.xlxs. In vivo STARR-seq activity for the 1,673 MACS2 regions identified in the focused STARR-seq plasmid library. Columns F and G indicate the 100 genomic DNA PCR-amplified regions used to prepare the STARR-seq plasmid library. Columns H-O show the raw sequencing reads in each STARR-seq RNA library, prepared from each liver after HDI, and Columns P-S show the raw sequencing reads obtained from the DNA library extracted from each liver, and from the original focused plasmid library. Columns T-AF show raw normalized reads per 10 million mapped reads, obtained by dividing the raw reads (columns) by the total number of sequencing reads in the corresponding sample. Columns AG-AN show the calculated enhancer activity of each region, based on the ratio of reads between the individual RNA library and the DNA library under the same condition. Columns AO-AQ indicate the average enhancer activity across the biological replicates for each condition.
Library strategy: STARR-seq
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| Submission date |
May 08, 2024 |
| Last update date |
Jun 17, 2024 |
| Contact name |
David J. Waxman |
| E-mail(s) |
djw@bu.edu
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| Organization name |
Boston University
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| Department |
Department of Biology and Bioinformatics Program
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| Street address |
5 Cummington Mall
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE267041 |
STARR-seq library profiling 100 defined open chromatin regions (‘focused library’) in livers of male and female mice treated with the nuclear receptor CAR (Nr1i3) agonist ligand TCPOBOP. |
| GSE267205 |
STARR-seq reporter activity of plasmid libraries delivered to mouse liver by hydrodynamic injection: In vivo MPRA assay for enhancer activity |
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| Relations |
| BioSample |
SAMN41267508 |
| SRA |
SRX24500022 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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