Computational protocol: ASXL2 is essential for haematopoiesis and acts as a haploinsufficient tumour suppressor in leukemia

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Protocol publication

[…] For ChIP-seq in murine cells 10 million BM c-Kit+ cells (isolated with anti–mouse CD117 microbeads (Miltenyi Biotec)) were used. For ChIP in SKNO-1 cells, 10 millions of GFP+ cell were used. Briefly, cells were fixed in a 1% methanol-free formaldehyde solution and then resuspended in sodium dodecyl sulfate (SDS) lysis buffer. Lysates were sonicated in an E220 focused-ultrasonicator (Covaris) to a desired fragment size distribution of 100–500 base pairs. IP reactions were performed with the indicated antibodies, each on approximately 500,000 cells. ChIP assays were processed on an SX-8G IP-STAR Compact Automated System (Diagenode) using a direct ChIP protocol as described elsewhere. Eluted chromatin fragments were then de-crosslinked and the DNA fragments purified using Agencourt AMPure XP beads (Beckman Coulter).Barcoded libraries were prepared from the ChIP-enriched and input DNA using a NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (New England BioLabs) and TruSeq Adaptors (Illumina) according to the manufacturer's instructions on an SX-8G IP-STAR Compact Automated System (Diagenode). Phusion High-Fidelity DNA Polymerase (New England BioLabs) and TruSeq PCR Primers (Illumina) were used to amplify the libraries, which were then purified to remove adaptor dimers using AMPure XP beads and multiplexed on the HiSeq 2000 (Illumina). Previosuly published anti-RUNX1 and AML1-ETO ChIP-seq were downloaded from the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE23730 (ref. ).Raw ChIP-seq data were analysed using Basepair software (http://www.basepairtech.com/) with pipelines including the following steps: the raw fastq data were trimmed using trim_galore to remove low-quality ends from reads (quality<15) and adapter sequences. The trimmed data was aligned using Bowtie2 (ref. ) to UCSC genome assembly hg19 (for human samples) or mm9 (for mouse samples). Duplicate reads were removed using Picard and bigwig files were created for visualization. Peaks were identified with Macs1.4 (ref. ) and transcription factor binding motifs were detected with Homer. Peaks overlapping with Satellite repeat regions were discarded and remaining filtered peaks were annotated using custom scripts based on UCSC refFlat data, where peaks between −2500, bp to 2500, bp of a transcription start site were marked as Promoter, the overlapping gene body were marked as Genebody and the rest were marked as Intergenic. For intergenic peaks, a gene was considered a target if it was within 1 Mb of the peak. All ChIP-seq reads were normalized and displayed as read counts per million mapped reads. […]

Pipeline specifications

Software tools Basepair, Trim Galore!, Bowtie2, Picard, HOMER
Databases GEO
Application ChIP-seq analysis
Organisms Homo sapiens, Drosophila melanogaster