2 protocols
normalization data transformation protocol
mRNA libraries were sequenced on Illumina genome analyzer and MIRA libraries were sequenced on Illumina HiSeq 2500 Sequenced reads of 51 bp that passed quality control filters(ASCII Qscore Offset 33) were used Sequence tags were mapped to the mouse genome (UCSC mm9 database based on NCBI Build 37 assembly) using the Bowtie2 We extended the 51-bp reads toward the 3’ end to cover DNA fragments bound by the MBD proteins (200 bp) We counted overlapping sequence tags at 50-bp resolution. We then calculated RPKM (reads per million base pairs mapped per kb) at 50-bp resolution The reads from mRNA-seq were aligned to mm9 using TopHat Genome_build: mm9
nucleic acid library construction protocol
Genomic DNA from 25-mg liver tissue samples was purified with a DNeasyR Blood & Tissue Kit. Total RNA from 3 livers collected from 3-month-old mice was extracted using Qiagen RNeasy mini kits. We generated cDNA libraries for sequencing using a Illumina mRNA sample prep kit (Hayward, CA). And TruSeq® DNA sample prep kit were used for generation of MIRA libraries.