title: GSM1631692: hMeDIP-seq Differentiated HL-60 Replicate-1; Homo sapiens; MeDIP-Seq
library_construction_protocol: Genomic DNA was isolated from three biological replicates of 3x10^6 differentiated (SYTOX Blue-, CD11b+) or undifferentiated (SYTOX Blue-, CD11b-) HL-60 cells using the DNeasy Blood & Tissue Kit (Qiagen, Inc.) following manufacturer’s instructions. 2.5 μg of the isolated genomic DNA from each biological replicate was resuspended in 130 μl Tris EDTA, pH 8.0, and sheared using a Covaris S2 instrument with the following settings: Duty Cycle: 10%, Intensity: 5, Cycles per Burst: 200 and Time: 240 seconds. The sheared DNA was purified by binding to an equal volume (~130 μl) of AMPure beads (Beckman Coulter, Inc.). This and all other AMPure purifications were performed as directed by the manufacturer. Libraries were constructed using the Illumina TruSeq DNA Sample Preparation Kit (Illumina, Inc. Cat# PE-940-2001), largely as directed by the "Low-Throughput" section of the kit manual but with the following changes; sheared DNA was blunted and extended by a single 3’ "A" using double the normal volumes of kit reagents. Each sample was divided in two parts (one half for MeDIP and the other half for hMeDIP) and subsequently 12 libraries were constructed. Each library was ligated to a different index-tag adapter (Index Adaptor). Instead of using the methylated adapters provided in the TruSeq kit, the sequences of TruSeq unique and Universal Adaptors were obtained from Illumina, Inc. and unmethylated versions of each adaptor were synthesized (IDT, Inc.) for use (sequences are available upon request from Illumina). The titers of the libraries, which ranged between 2.9 and 7.33 nM, were determined in duplicate by qPCR on a StepOne (Applied Biosystems, Inc.) instrument and a SYBR-Green Library Quantification Kit - Illumina (Kapa Biosystems, Inc.). 40 µl of library adapter ligated DNAs was subjected to methylated and hydroxymethylated DNA immunoprecipitation using kits from Diagenode, Inc. (meDIP: mc-magme-048; hmeDIP: AF-110-0016), following the manufacturer’s instructions.
attributes.tag: GEO Accession
attributes.value: GSM1631692