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| Status |
Public on Jun 13, 2023 |
| Title |
CH12_PROseq_shLacZ_rep1 |
| Sample type |
SRA |
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| Source name |
CH12
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| Organism |
Mus musculus |
| Characteristics |
cell type: CH12
genotype: shLacZ
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| Growth protocol |
CH12 cells cultured in complete RPMI medium wit IL4/CD40/TGF beta stimulation,primary B cells were isolated from C57BL/6 (WT/WT) mice spleens using anti-CD43 MicroBeads (Miltenyi Biotec) and expanded in complete RPMI containing 5 µg/ml LPS (Sigma-Aldrich) and 5 ng/ml mouse recombinant IL-4 (Sigma-Aldrich) to allow B cell activation and class switch recombination to IgG1. B cells were harvested 72h after activation and 4 x 10^7 cells
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| Extracted molecule |
total RNA |
| Extraction protocol |
trizol extraction
PRO-seq was performed as described previously with minor modifications. To isolate nuclei, CH12 cells and Drosphila S2 cells were resuspended in cold Buffer IA (160mM sucrose, 10mM Tris-Cl pH 8, 3mM CaCl2, 2mM MgAc2, 0.5% NP-40, 1mM DTT added fresh), incubated on ice for 3 min and centrifuged at 700g for 5 min. The pellet was resuspended in nuclei resuspension buffer NRB (50mM Tris-Cl pH 8, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). For each run-on, 10 million CH12 nuclei were spiked with 10% Drosophila S2 nuclei in a total of 100µL NRB and incubated at 30°C for 3 min with 100 µL 2x NRO buffer including 5µl of each 1mM NTP (biotinylated ATP and GTP, and unlabelled UTP and CTP). The following steps were performed as described62 with the following changes: (1) we used different adapters, namely, 3' RNA adapter 5Phos/NNNNNNNGAUCGUCGGACUGUAGAACUCUGAAC/3InvdT-3’ and 5’RNA adapter: 5’-CCUUGGCACCCGAGAAUUCCANNNN-3’. (2) 3’ and 5’ ligations which were done at 16°C overnight, and (3) CapClip pyrophosphatase (Cellscript) used for 5’ decapping. RNA was reverse transcribed by SuperScript III RT (Invitrogen) with RP1 Illumina primer to generate cDNA libraries. Libraries were amplified using barcoding Illumina RPI-x primers and the universal RP1 and KAPA HiFi Real-Time PCR Library Amplification Kit. Amplified libraries were subjected to electrophoresis on 2.5% low melting agarose gel and amplicons from 150-350 bp were extracted from the gel, multiplexed and sequenced on Illumina platform NextSeq 550 SR75 high. Bioinformatics analyses were performed as described62 but additionally the random 8-mer was used to exclude PCR duplicates and only deduplicated reads were aligned.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
PRO-seq
CH12_PROseq_shLacZ_20kb.bw
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| Data processing |
library strategy: PRO-seq
3' adaptors were removed using cutadapt v1.4.2 with -O 5 -a NNNNTGGAATTCTCGGTGCC) and 9 nucleotides from their 5′ were removed and subsequently reverse complemented using fastx_reverse_complement (http://hannonlab.cshl.edu/fastx_toolkit; version 0.0.13)
Trimmed reads longer than 18bp were aligned to a hybrid mouse drosophila genome (mouse genome build NCBI m37, GCA_000001635.18, drosophila FlyBase release 5) using bowtie v1.0.0 with -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 v2.27.1 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 were generated with deeptools bamCoverage v2.1.0 by applying 1,000,000/count_of_Spike as scaling factor
Assembly: mm9
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| Submission date |
Apr 03, 2023 |
| Last update date |
Jun 13, 2023 |
| Contact name |
Tobias Neumann |
| Organization name |
IMP
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| Street address |
Campus-Vienna-Biocenter 1
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE228877 |
RIF1 regulates replication origin activity and early replication timing in B cells [PRO-seq] |
| GSE228880 |
RIF1 regulates early replication timing in murine B cells. |
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| Relations |
| BioSample |
SAMN34060507 |
| SRA |
SRX19857756 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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