title: GSM2942302: gm_5ng_rep1; Homo sapiens; MeDIP-Seq
library_construction_protocol: Hemizygous transgenic female mice (FVB-Tg(C3-1-TAg)cJeg/JegJ, Jackson Laboratory) were used in this study. Mice were euthanized at age of 6, 16, 23 weeks by exposing mice to 100% carbon dioxide at 1 – 2 l/min for 5 min followed by cervical dislocation. Mice were sprayed with 70% ethanol on the ventral side. Skin on the ventral side was cut through, pulled away from mouse body, and pinned down to expose mammary glands. Mammary tumors were identified and cut off. The size and weight of a tumor were recorded.Genomic DNA from GM12878 cells was extracted from 106 cells using Blood & Cell Culture DNA Mini Kit (Qiagen). Mouse genomic DNA was extracted from mouse mammary tissues using DNeasy Blood & Tissue Kit (Qiagen). Extracted genomic DNA was sonicated with a Covaris E220 sonicator for 180 s with 10 % duty cycle, 50 peak incident power and 200 cycles per burst. The concentration of sonicated DNA was quantified using a Qubit 2.0 fluorometer with dsDNA HS Assay kit (Q32851, Life Technologies). Different sample sizes (0.5~100 ng) were aliquoted and diluted with MeDIP buffer to generate a final volume of 50 µl for microfluidic MeDIP. 0.5 ng, 5 ng, 10 ng, or 100 ng of the sonicated sample was used as the input. Sonicated DNA samples were freshly denatured into single strand DNA (ssDNA) under 95℃ for 15 min and put on ice for 5 min before microfluidic MeDIP. Sequencing libraries were prepared by DNA SMART™ ChIP-Seq kit (634865, Clontech). This kit generates sequencing libraries from low-input ssDNA MeDIP samples (100 pg - 10 ng) without using ligation. Sequencing libraries were amplified by PCR using primers containing Illumina adapters. The libraries were purified using Ampure XP beads (A63880, Beckman Coulter). The library fragment size was determined using high sensitivity DNA analysis kit (5067-4626, Agilent) on an Agilent 2200 TapeStation. Sonicated DNA for MeDIP had average fragment size around 200 bp. After library preparation, the average peak size shifted to 350 bp as library construction added about 150 bp adaptor (75 bp on each end) to the MeDIP DNA. KAPA library quantification kit (KK4809, Kapa Biosystems) was used to determine effective library concentrations. The final concentrations of libraries submitted for sequencing were ~8 nM. The libraries were sequenced on an Illumina HiSeq 4000 with single-end 50 nt read. Typically 15-20 million reads were generated per library.
attributes.tag: GEO Accession
attributes.value: GSM2942302