title: Absence of dosage compensation and the arrested stage of sex chromosome evolution in ostriches: BM2
design: Total RNA was extracted with RNeasy Mini Kit/Lipid Tissue Mini Kit (Qiagen) following the kit protocols.     Illumina HiSeq sequencing: Sequencing libraries were prepared from 1-4µg of total RNA according to the TruSeq RNA sample preparation guide #15008136 revA using reagents from the TruSeq RNA sample prep kit set A and set B v1 (Illumina, San Diego, CA). Briefly, poly-A containing mRNA were purified from 1.5µg of total RNA using poly-T oligo-attached magnetic beads, followed by fragmentation of the mRNA. First strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexamers, followed by second strand synthesis according to the manufacturer reagents and protocols. The overhangs on the DNA fragments were end-repaired followed by purification using AMPure XP beads (Beckman Coulter, Brea, CA). An A-base was added to the blunt ends of the DNA fragments and adapters and indextags for sequencing was ligated, followed by purification using AMPure XP beads. The library was amplified for 12-15 PCR cycles, followed by purification using AMPure XP beads. The quality of the library was evaluated using the Agilent Technologies 2100 Bioanalyzer and a DNA 1000-kit.  The adapter-ligated fragments were quantified by qPCR using the Library quantification kit for Illumina (KAPA Biosystems, Cambridge, MA) on a StepOnePlus instrument (Applied Biosystems/Life technologies, Carlsbad, CA) prior to cluster generation and sequencing. A 6-10 pM solution of the pooled libraries (see below) was subjected to cluster generation on the cBot instrument (Illumina Inc.). Paired-end sequencing was performed for 100 cycles in one lane using a HiSeq2000 instrument (Illumina Inc), according to the manufacturer’s protocols.   Base calling was done on the instrument by RTA 1.10.36 and the resulting .bcl files were converted to Illumina qseq format with tools provided by OLB-1.9.0 (Illumina Inc.). To separate samples and PhiX control DNA sequenced in the same lane as the sample libraries, the qseq-files were demultiplexed, allowing for one mismatch. Both demultiplexing and mapping were done with CASAVA 1.7.0 (Illumina Inc.). Additional statistics on sequence quality were compiled from the base call files with an in-house script.    Sequencing was performed by the SNP&SEQ Technology Platform in Uppsala, Sweden www.sequencing.se.