title: Illumina HiSeq 4000 sequencing; A Nucleosome Destabilizing Factor that Facilitates Transcription in Chromatin
design: A Nucleosome Destabilizing Factor that Facilitates Transcription in Chromatin
library_construction_protocol: Human cells were cultured and collected by following a standard protocol. For mouse bone marrow-derived macrophages (BMDM), bone marrow was isolated from 8- to-10-week-old C57Bl/6 (Harlan Laboratories) female mice. BMDM were generated in DMEM supplemented with M-CSF for 7 days and activated with Kdo2-Lipid A (KLA) for 6 hours as described in Kaikkonen, M. U. et al. Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol. Cell 51, 310-325. Antibodies against H3K27me3 (07-449) were from Millipore Human Cells were maintained in DMEM with 10% FBS, 5% CO2, 37oC. Mouse BMDMs were generated from bone marrow and cultured in DMEM supplemented with M-CSF for 7 days and activated with KLA as descried.  Mouse BMDM were treated with 100 ng/ml of Kdo2-Lipid A as indicated. For human ChIP-seq, ~15 million wild-type HeLa S3 cells or the indicated hNDF (NRG1) knockout cells were collected and crosslinked with 1% formaldehyde. After cell lysis and genome fragmentation by sonication, samples were incubated with 20 μL of Dynabeads Protein A that were pre-bound with 1 μL of NDF antiserum. [Note that 1 μL of hNDF antiserum was optimal for hNDF antiserum #7. For hNDF antiserum #6, 2 μL was the optimal volume.] Bound DNA were isolated with MinElute columns (Qiagen). For human RNA-seq, total RNA was purified by using the PureLink RNA Mini RNA Kit (Thermo Fisher Invitrogen). Mouse BMDM ChIP-seq and RNA-seq were performed as described in Kaikkonen, M. U. et al. Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol. Cell 51, 310-325.  Human ChIP-seq libraries were prepared by end-repair, A-tailing, and ligation of Illumina TruSeq adaptors, which was then followed by PCR amplification and size selection with AMPure beads (Beckman Coulter). Human RNA-seq libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina). Mouse ChIP-seq libraries were prepared from ChIP DNA and inputs by blunting, A-tailing, and adapter ligations were performed by using NextFlex barcode adapters. Mouse RNA-seq libraries were prepared from poly(A)-enriched mRNA, as described in Kaikkonen, M. U. et al. Mol. Cell 51, 310-325.
attributes.tag: Experimental Factor: stimulus
attributes.value: Kdo2-Lipid A, 100 nanogram per milliliter
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: H3K36me3