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Sample GSM5288412 Query DataSets for GSM5288412
Status Public on May 31, 2021
Title BN_MB6_G
Sample type SRA
 
Source name Brain
Organism Mus musculus
Characteristics strain: C57BL/6 x Castaneus F1 hybrid
tissue: Brain
age: post natal day 24
gender: male
Extracted molecule total RNA
Extraction protocol ChRO-seq library preparation: ChRO-seq libraries were prepared following a recent protocol (Mahat et al., 2016). We prepared some libraries to achieve single nucleotide resolution for the Pol II active site. In these cases, the chromatin pellet was incubated with 2x run-on buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2,1 mM DTT, 300 uM KCl, 20 uM  Biotin-11-ATP (Perkin Elmer, NEL544001EA), 200 uM Biotin-11-CTP (Perkin Elmer, NEL542001EA), 20 uM Biotin-11-GTP (Perkin Elmer, NEL545001EA), 200 uM Biotin-11-UTP (Perkin Elmer, NEL543001EA)) for 5 minutes at 37 C. In some libraries we modified the run-on buffer to extend the length of reads for more accurate allelic mapping at the expense of single nucleotide resolution for the Pol II active site. In these cases, the run-on reaction was performed using a different ribonucleotide composition in the nuclear run-on buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2,1 mM DTT, 300 mM KCl, 200 μ M  ATP (New England Biolabs (NEB), N0450S), 200 μ M  UTP, 0.4 μ M CTP, 20 μ  M Biotin-11-CTP (Perkin Elmer, NEL542001EA), 200 μ  M GTP (NEB, N0450S)).The run-on reaction was stopped by adding Trizol LS (Life Technologies, 10296-010) and RNA was pelleted with the addition of GlycoBlue (Ambion, AM9515) to visualize the RNA. RNA pellet was resuspended in diethylpyrocarbonate (DEPC)-treated water. RNA was heat denatured at 65 C for 40 s to remove secondary structure. RNA was fragmented using base hydrolysis (0.2N NaOH on ice for 4 min). RNA was purified using streptavidin beads (NEB, S1421S) and removed from beads using Trizol (Life Technologies, 15596-026). We ligated a 3′ adapter ligation using T4 RNA Ligase 1 (NEB, M0204L). We performed a second bead binding followed by a 5′ decapping with RppH (NEB, M0356S). RNA was phosphorylated on the 5’ end using T4 polynucleotide kinase (NEB, M0201L) then ligated onto a 5’ adapter. A third bead binding was then performed. The RNA was then reverse transcribed using Superscript III Reverse Transcriptase (Life Technologies, 18080-044) and amplified using Q5 High-Fidelity DNA Polymerase (NEB, M0491L) to generate the ChRO-seq libraries. Libraries were sequenced using an Illumina HiSeq by Novogene.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 4000
 
Description nascent RNA
pooled sample G
BN_MB6_all_R1.B6.bowtie.gz
BN_MB6_all_R1.CAST.bowtie.gz
BN_MB6_all_R1.B6.specific.5prime.1bp.map2ref_plus.bw
BN_MB6_all_R1.B6.specific.5prime.1bp.map2ref_minus.bw
BN_MB6_all_R1.CAST.specific.5prime.1bp.map2ref_plus.bw
BN_MB6_all_R1.CAST.specific.5prime.1bp.map2ref_minus.bw
BN_MB6_all_R1.identical.5prime.1bp.map2ref_plus.bw
BN_MB6_all_R1.identical.5prime.1bp.map2ref_minus.bw
Data processing Library strategy: ChRO-Seq
For ChRO-seq reads, adaptor sequences were trimmed with the cutadapt 1.16. PCR duplicates were removed using unique molecular identifiers (UMIs) in the sequencing data with prinseq-lite.pl. The reads were seperated with a custom J-barcode. The processed reads were then aligned with bowtie to to the diploid genome as input for AlleleDB.
The first base of nascent RNA in B6-specific, CAST-specific, or identical reads without SNPs was liftover to ref genome (mm10), and shown as bigwig files
Genome_build: mm10
Supplementary_files_format_and_content: ChRO-seq data are provided in standard bigWig file formats.
 
Submission date May 10, 2021
Last update date May 31, 2021
Contact name Charles G. Danko
E-mail(s) dankoc@gmail.com
Organization name Cornell University
Department Baker Institute for Animal Health, College of Veterinary Medicine
Street address Hungerford Hill Rd.
City Ithaca
State/province NY
ZIP/Postal code 14853
Country USA
 
Platform ID GPL21103
Series (1)
GSE174171 Genetic Dissection of the RNA Polymerase II Transcription Cycle with ChRO-seq from F1 hybrid mice
Relations
BioSample SAMN19093442
SRA SRX10828660

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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