4 protocols
normalization data transformation protocol
HiSeq Control Software v2.0.5 and Casava v1.8.2 were used for basecalling and de-multiplexing. ChIP-seq reads were aligned to the dm3 genome assembly (FlyBase 5.22) using Bowtie 0.12.7 with parameters -t -p 4 -a -m 1 --maxins 900 --tryhard --best --strata -S --chunkmbs 512. Sequence tags multiply matched to repetitive sequences were excluded in all ChIP-seq and RNA-seq results. Sequence alignment information was further processed by SAMtools v0.1.18 (Li et al., 2009) to generate a pileup of read bases which was used for obtaining the genome-wide profiling data format by python scripts (SGR format with 25bp bin) compatible with the Integrated Genome Browser (Affymetrix). Normalization between different culture conditions (25˚C and 31˚C) was done by calculating the correction factors based on ChIP-qPCR. At least five regions were used for the normalization. Genome_build: dm3 Supplementary_files_format_and_content: sgr files with 25bp bin size were generated from a pileup of read bases using samtools v0.1.18 mpileup and python scripts; Scores represent qPCR-normalized number of reads per 25bp bin.
nucleic acid library construction protocol
Live cells were crosslinked with 1.8% formaldehyde for 10 min at 25°C and soluble chromatin prepared as described in Schwartz et al., 2006. Libraries were prepared according to Illumina's instructions from TruSeq ChIP Sample Preparation Kit with some modifications. Indexed adaptors from TruSeq ChIP Sample Prep Kit were used for barcoding. After adapter ligation, 250-500bp size was selected by gel size selection, and the subsequent amplification was done with 15 cycles. With the constructed libraries, 100bp paired-end sequencing was performed by Illumina HiSeq technology
sample treatment protocol
EZ2-2 cells were grown at 25°C or 31°C for 12 days.
growth protocol
EZ2-2 cells were grown in Schneider's medium (Gibco) supplemented with 10% FBS, 100U/ml of Penicillin G, 100ug/ml of Streptomycin sulfate and 292 ug/ml of L-glutamine.