title: GSM3386918: CD16_iPSC_8801; Homo sapiens; RNA-Seq
library_construction_protocol: ChIPmentation:The purified cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature at a concentration of 15x106 cells/ml. The fixed cells were sonicated for 5 minutes (30 sec on; 30 sec off) using the Diagenode Bioruptor and centrifuged at maximum speed for 10 minutes. Chromatin of 2x105 cells was incubated overnight in dilution buffer (167mM NaCl, 16,7 mM Tris (pH 8), 1,2mM EDTA, 1% Triton X-100) with 1 µg H3K4me3 antibody at 4C. ProtA/G beads were blocked with dilution buffer (+0,15% SDS) and incubated with the chromatin-Ab for one hour at 4C. Beads were washed with three different wash buffers and the DNA library was prepared using the Nextera DNA sample prep kit  ChIP-Seq: Control and AML1-ETO iMonocyte and iGranulocyte suspensions were crosslinked with 1% formaldehyde for 10 minutes at room temperature at a concentration of 15x106 cells/ml. The fixed cells were sonicated for 4 minutes (30 sec on; 30 sec off) using the Diagnode Bioruptor and centrifuged at maximum speed for 10 minutes. Chromatin of about 5x105 cells was incubated overnight in dilution buffer (167mM NaCl, 16,7 mM Tris (pH 8), 1,2mM EDTA, 1% Triton X-100) with 1 µg H3K27ac antibody at 4C. ProtA/G beads were blocked and incubated with the chromatin-Ab for one hour at 4C. Beads were washed with three different wash buffers and chromatin was eluted from the beads. DNA-proteins were de-crosslinked (200mM NaCl and 4 µl proteinase K (10mg/ml)), by incubation for four hours at 65C and samples were purified using the Qiaquick MinElute PCR purification kit according manufacturer's protocol. The procedure was performed according the Blueprint Histone ChIP protocol RNA-Seq:imonocytes and igranulocytes were purified by MACS sort using CD14 and CD16 beads (Miltenyi Biotec). Total RNA was isolated from 1x106 purified CD14+ imonocytes and CD16+ igranulocytes as well as from control and AML1-ETO imonocytes and igranulocytes using the RNeasy mini kit (Qiagen) and performing on-column DNaseI treatment. Ribosomal RNA was depleted from 500ng of RNA by using the ribo-zero rRNA removal kit according manufacturer's protocol (Illumina) Libraries were generated from precipitated DNA of 5 million cells using the Kapa hyper prep kit. End repair and A-tailing was performed on the double strand DNA using end repair and A-tailing buffer. Subsequently, the adapters were ligated and a post-ligation cleanup was performed using Agencourt AMPure XP reagent. The libraries were amplified by PCR using the Kapa Hifi hotstart readymix and primer mix for 10 cycles. Samples were purified using the Qiaquick MinElute PCR purification kit and 300 bp fragments were selected from E-gel and the size was checked on the agilent bioanalyzer. Samples were sequenced on the Illumina HiSeq 2000, generating 42 bp single end reads.
attributes.tag: GEO Accession
attributes.value: GSM3386918