Computational protocol: Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes

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Protocol publication

[…] 10 ng, as quantified by Qubit dsDNA HS Assay Kit (Life Technologies), of input and of immuno-precipitated-material was used for library preparation. For one ChIP-Seq experiment chromatin prepared from 100 × 106 HeLa-LTR-Luciferase cells was incubated with 10 μg of antibody (the same protocol was used for all antibodies except for antibodies against histone modifications for which chromatin of only 50 × 106 HeLa-LTR-Luciferase cells was used). Under these conditions the following total amounts of DNA were precipitated:An amount of 10 μg of anti-RNAPII total (Santa Cruz, sc-899): Replicate A: 53.7 ng. Replicate B: 71.1 ng.An amount of 5 μg of anti-H3K4me3(Abcam, ab8580): Replicate A: 1290, ng. Replicate B: 1251, ng.An amount of 10 μg of anti-NELF-E (Millipore, ABE48): Replicate A: 21 ng. Replicate B: 27 ng.An amount of 10 μg of anti-Spt5 (BD Transduction Laboratories, 611106): Replicate A: 18 ng. Replicate B: 24 ng.10 μg of anti-INTS3 (Abcam, ab70451): Replicate A: 15 ng. Replicate B: 15.9 ng.An amount of 10 μg of anti-INTS3 (Bethyl Laboratories, A302-051A): Replicate A: 81 ng. Replicate B: 81 ng.An amount of 10 μg of anti-INTS3 (Proteintech, 16620-1-AP): Replicate A: 109 ng. Replicate B: 108.3 ng.An amount of 10 μg of anti-INTS11 (Bethyl Laboratories, A301-274A): Replicate A: 10.8 ng. Replicate B: 13.2 ng. 10 μg of anti-INTS13 (Bethyl Laboratories, A303-575A): Replicate A: 33 ng. Replicate B: 43.5 ng. 10 μg of anti-INTS13 (Proteintech, 19892-1-AP): Replicate A: 45 ng. Replicate B: 55.2 ng.Libraries were constructed using either Illumina ChIP-Seq DNA Sample Prep Kit (non-multiplexed libraries) or TruSeq ChIP Sample Prep Kit (multiplexed libraries) according to manufacturer’s instructions. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Data quality was assessed using fastqc from the Babraham Institute and the software SAV (Sequence Analysis Viewer) (Illumina). De-multiplexing and alignment were performed using Illumina's sequencing analysis software (CASAVA 1.8.2) (Illumina). […]

Pipeline specifications

Software tools HSC, FastQC, BaseSpace
Application ChIP-seq analysis
Organisms Human immunodeficiency virus 1
Diseases Immunologic Deficiency Syndromes