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| Status |
Public on Jan 20, 2023 |
| Title |
priB_PROseq_rest_rep2 |
| Sample type |
SRA |
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| Source name |
primary B-cells
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| Organism |
Mus musculus |
| Characteristics |
treatment: resting
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| Growth protocol |
Complete RPMI medium with LPS and IL4 for activation
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| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate nuclei, murine and Drosphila S2 cells were resuspended in cold Buffer IA (160 mM Sucrose, 10 mM Tris-Cl pH 8, 3 mM CaCl2, 2 mM MgAc2, 0.5% NP-40, 1 mM DTT added fresh), incubated on ice for 3 min and centrifuged at 700 g for 5 min. The pellet was resuspended in nuclei resuspension buffer NRB (50 mM Tris-Cl pH 8, 40% Glycerol, 5 mM MgCl2, 0.1 mM EDTA). For each run-on, 107 nuclei of the sample and 10% Drosophila S2 nuclei were combined in a total of 100 µL NRB and incubated at 30°C for 3 min with 100 µL 2x NRO buffer including 5µl of each 1mM NTPs. Reactions were terminated with TRIzol and RNA extracted as per the manufacturer's protocol.
3’ ligation was performed at 16°C overnight with adapter 5’5Phos/NNNNNNNGAUCGUCGGACUGUAGAACUCUGAAC/3InvdT-3’. RNA was reverse transcribed by SuperScript III RT with RP1 Illumina primer to generate cDNA libraries. Libraries were amplified with barcoding Illumina RPI-x primers and the universal forward primer RP1 using KAPA HiFi Real-Time PCR Library Amplification Kit. Amplified libraries were subjected to gel electrophoresis on 2.5% low melting agarose gel and amplicons from 150 to 350 bp were extracted from the gel, multiplexed and sequenced on Illumina platform NextSeq 550 SR75.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
nascent RNA
PRO-seq
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| Data processing |
Library strategy: PRO-seq
PRO-seq
Identical reads were collapsed using sort -k 10,10 -u.
The 3' adaptors were removed using cutadapt (v1.4.2 Read1: cutadapt -O 5 -a NNNNTGGAATTCTCGGTGCC ) and 9 nucleotides from their 5′ were removed.
The trimmed reads were reverse complemented using fastx_reverse_complement (http://hannonlab.cshl.edu/fastx_toolkit ).
Trimmed reads >18 were aligned using bowtie (v 1.0.0, -v 2 --best --strata --tryhard -m 1 --chunkmbs 256).
Unique mappers from the resulting BAM file were used to create unstranded, stranded and reverse stranded coverage tracks for visualisation using bedtools (v2.27, bedtools genomecov -split -bg, bedtools genomecov -split -bg -strand '+', bedtools genomecov -split -bg -strand '-') and bedGraphToBigWig from the kent-tools (bedGraphToBigWig v4, bedGraphToBigWig -blockSize=256 -itemsPerSlot=1024).
Normalised coverage tracks were created by dividing the coverage at each position with the total number of aligned reads per million.
Spike-in normalised coverage tracks of the 3’ position were created using bedtools genomecov (v2.27, bedtools genomecov -3 -bg -scale, bedtools genomecov -3 -bg -scale '+', bedtools genomecov -3 -bg -scale '-‘)
Assembly: hybrid mouse drosophila genome (GRCm38 and drosophila FlyBase release 5)
Supplementary files format and content: bigWigs of rpm-normalized and spike-in normalized read densities
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| Submission date |
Mar 09, 2022 |
| Last update date |
Jan 23, 2023 |
| Contact name |
Maximilian Christian von der Linde |
| E-mail(s) |
maximilian.linde@imp.ac.at, max_vdl@outlook.com
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| Phone |
+4368181646556
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| Organization name |
Institute of Molecular Pathology
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| Lab |
Pavri GRP
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| Street address |
Campus-vienna-biocenter 1, IMP, Pavri GRP
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| City |
Vienna |
| State/province |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE198180 |
A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination [PRO-seq] |
| GSE198182 |
A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination |
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| Relations |
| BioSample |
SAMN26537414 |
| SRA |
SRX14412950 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5940184_priB_PROseq_rest_rep2_fwd_rpm.bw |
113.0 Mb |
(ftp)(http) |
BW |
| GSM5940184_priB_PROseq_rest_rep2_fwd_spikenorm.bw |
54.9 Mb |
(ftp)(http) |
BW |
| GSM5940184_priB_PROseq_rest_rep2_rev_rpm.bw |
111.8 Mb |
(ftp)(http) |
BW |
| GSM5940184_priB_PROseq_rest_rep2_rev_spikenorm.bw |
54.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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