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Sample GSM5288413

Query DataSets for GSM5288413

Status

Public on May 31, 2021

Title

BN_PB6_B

Sample type

SRA

Source name

Brain

Organism

Mus musculus

Characteristics

strain: Castaneus x C57BL/6 F1 hybrid
tissue: Brain
age: post natal day 25
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 B
BN_PB6_all_R1.B6.bowtie.gz
BN_PB6_all_R1.CAST.bowtie.gz
BN_PB6_all_R1.B6.specific.5prime.1bp.map2ref_plus.bw
BN_PB6_all_R1.B6.specific.5prime.1bp.map2ref_minus.bw
BN_PB6_all_R1.CAST.specific.5prime.1bp.map2ref_plus.bw
BN_PB6_all_R1.CAST.specific.5prime.1bp.map2ref_minus.bw
BN_PB6_all_R1.identical.5prime.1bp.map2ref_plus.bw
BN_PB6_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