title: GSM5099800: Sumo2-3_Day3_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were lysed, homogenized and collected using 1 ml of RNAzol®RT before adding 0.4 ml of water per 1 ml of homogenate. The mixture was vortexed for 5 min and centrifuged at 12,000 g for 15 min. Following centrifugation, DNA, proteins and polysaccharides form a semisolid pellet at the bottom of the tube. The RNA remains soluble in the supernatant. The supernatant was transferred to a new tube and 1 volume of 70% ethanol was added. The mixture was homogenized by pipetting. Samples were then applied to a RNeasy spin column and RNAs were purified according to Qiagen's instructions. Base conversion was performed using the Quant-seq 3' mRNA-Seq library preparation kit. ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). ChIP-seq libraries were prepared from 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7 cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.