ChIP-seq analysis of H3K4me3 in plasma cells

As a complementary approach to identify variants located in genomic regions with regulatory activity in plasma cells, we analyzed previously published^{31 (link)} chromatin immunoprecipitation sequencing (ChIP-seq) data for the H3K4me3 histone modification^{38 (link)}. Briefly, L363 cells (DSMZ) were cross-linked with 1% paraformaldehyde (ThermoFisher, #28908). DNA was sonicated into 200–400 bp fragments (Bioruptor Pico Sonication System, Diagenode, Belgium). For pull-down, we used 1–10 μg of H3K4me3 antibody (Millipore, #04-745). Fragments were de-cross-linked and purified (Zymogen, #D5205). ChIP-seq libraries were prepared using the ThruPLEX DNA-seq Kit (Rubicon Genomics, #R400406) and sequenced on Illumina HiSeq 2500 sequencer (paired-end; 2 × 125 cycles). De-multiplexing and generation of FASTQ files was performed using bcl2fastq v.1.8. FastQC (v0.11.5)^{39 (link)} was used to assess read quality low-quality bases were removed using Trimmomatic (v.0.36)^{40 (link),41 (link)} prior to alignment. using Bowtie2 (v.2.3.0)^{41 (link)}. Coverage in 50 bp over the SOHL2 region was calculated with the GenomicAlignments and GenomicRanges R-packages⁴² (coverage and binnedAverage functions) and scaled to Counts-per-million (CPM) relative to the total number of reads per library.

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Duran-Lozano L., Thorleifsson G., Lopez de Lapuente Portilla A., Niroula A., Went M., Thodberg M., Pertesi M., Ajore R., Cafaro C., Olason P.I., Stefansdottir L., Bragi Walters G., Halldorsson G.H., Turesson I., Kaiser M.F., Weinhold N., Abildgaard N., Andersen N.F., Mellqvist U.H., Waage A., Juul-Vangsted A., Thorsteinsdottir U., Hansson M., Houlston R., Rafnar T., Stefansson K, & Nilsson B. (2021). Germline variants at SOHLH2 influence multiple myeloma risk. Blood Cancer Journal, 11(4), 76.

Publication 2021

paraformaldehyde Bioruptor Pico Sonication System H3K4me3 antibody ThruPLEX DNA-seq Kit HiSeq 2500 sequencer

Antibody Bp 400 Cells Genomic H3k4me3 Histone Library Paraformaldehyde Plasma cells Pull Zymogen

Corresponding Organization :

Other organizations : Lund University, deCODE Genetics (Iceland), Institute of Cancer Research, Heidelberg University, University Hospital Heidelberg, Odense University Hospital, University of Southern Denmark, Aarhus University Hospital, Södra Älvsborg Hospital, Norwegian University of Science and Technology, Rigshospitalet, University of Iceland, Broad Institute

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Coverage in 50 bp over the SOHL2 region, scaled to Counts-per-million (CPM) relative to the total number of reads per library

control variables

L363 cells (DSMZ) were used
Cells were cross-linked with 1% paraformaldehyde (ThermoFisher, #28908)
DNA was sonicated into 200–400 bp fragments (Bioruptor Pico Sonication System, Diagenode, Belgium)
1–10 μg of H3K4me3 antibody (Millipore, #04-745) was used for pull-down
Fragments were de-cross-linked and purified (Zymogen, #D5205)
ChIP-seq libraries were prepared using the ThruPLEX DNA-seq Kit (Rubicon Genomics, #R400406)
Sequencing was performed on Illumina HiSeq 2500 sequencer (paired-end; 2 × 125 cycles)
De-multiplexing and generation of FASTQ files was performed using bcl2fastq v.1.8
FastQC (v0.11.5) was used to assess read quality
Low-quality bases were removed using Trimmomatic (v.0.36)
Reads were aligned using Bowtie2 (v.2.3.0)

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