title: 1C2M1 (cold shock, 0C, replicate 1)
design: RNA Isolation and quality verification  Total RNA from 10 Drosophila imagoes was extracted using miRNeasy Mini Kit (Qiagen, Germany) by following the manufacturer's instructions. Fluorescence quantitation was performed with Qubit® 2.0 Fluorometer (Invitrogen, USA). The quality of the total RNA was verified on Agilent 2100 Bioanalyzer using Agilent RNA 6000 Nano chip (Agilent Technologies, USA). RNA Integrity Number (RIN) value was ≥ 8 for each sample.     Total RNA sequencing sample preparation  Illumina TruSeq™ RNA Sample Preparation Kit (Low-Throughput protocol)  was used to prepare samples for the mRNA sequencing libraries . In summary, 2.5–3.0 µg of total RNA from each sample was used to purify the poly-A containing mRNA molecules by poly-T oligo-attached magnetic beads, with two rounds of purification. During the second elution of poly-A RNA, the RNA was also fragmented and primed for cDNA synthesis according to the manufacturer’s protocol. cDNA synthesis from fragmented RNA and further cDNA conversion into double-stranded (ds) cDNA were performed by using a SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, USA). Ampure XP beads were used to separate ds cDNA from the second-strand reaction mix. The double-stranded cDNA was subjected to library preparation using the Illumina TruSeq™ RNA Sample Preparation Kit (Low-Throughput protocol) according to the manufacturer’s protocol (Illumina, USA).    Preparation of cDNA library  The construction of a cDNA library was performed according to the manufacturer's instructions using TruSeq™ RNA Sample Preparation Kit (Illumina, USA). 2.5 µg of high-quality RNA from each sample was used in purify and fragment mRNA procedures. The cleaved and primed mRNA fragments in turn were subjected to first-strand and second-strand cDNA synthesis. The generated double-stranded cDNA (ds cDNA) was further subjected to conversion the overhangs resulting from fragmentation into blunt ends per the manufacturer's instructions. A single “A” nucleotide was added to the 3’-terminal ends of blunt fragments through adenylation reaction to prevent the ligating of them to one another during adapter ligation process. Then the multiple indexing adapters containing single “T” nucleotide on the 3’ end were complementarity ligated to the ends of the adenylated ds cDNA to require for hybridization of cDNA library onto a flow cell. The in-line controls for tracking the steps involved in converting ds cDNA into libraries were used. Selective enrichment of cDNA fragments that have adapters ligated on both ends was performed using Illumina PCR Primer Cocktail in 11 cycle PCR reaction according to manufacturer's protocol. The library fragments were purified with AMPure XP system (Beckman Coulter, USA) after each step of enzymatic reaction.    Library validation  Library quantification was performed using a qPCR method as described. The quality of libraries was checked on Agilent 2100 Bioanalyzer using High Sensitivity DNA chip (Agilent Technologies, USA). The final library products were bands at approximately 260 bp.    Sequencing procedure  Indexed cDNA libraries were normalized to 2nM and pooled together in equal volumes. The clustering of pooled cDNA library was performed on a cBot Cluster Generation System (Illumina, USA) according to the vendor’s instructions. After cluster generation, the cDNA library was sequenced on a HiSeq™2000 platform (Illumina, USA) and 50 bp single reads were generated. Raw sequencing reads were obtained using Illumina HiSeq Analysis Software and stored in FASTQ format.
identifiers.namespace: Moscow Institute of Physics and Technology (State
identifiers.id: 1C2M1 (cold shock, 0C, replicate 1)