title: GSM3753206: LS_T2; Saccharum hybrid cultivar; RNA-Seq
library_construction_protocol: Total RNA was extracted from frozen samples using the Qiagen RNA plant mini-kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The quality and quantity of RNA were determined by Nanodrop and Qubit, respectively, and Agilent 2100 was used to evaluate the RNA Integrity. High quality RNA samples with Integrity Number >8 were used for constructing cDNA libraries and sequencing using Illumina HiSeq 2000 platform (Beijing Novogene Bioinformatics Technology Co. Ltd., Beijing, China). A total amount of 3μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using Illumina TruseqTM RNA sample prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37oC for 15 min followed by 5 min at 95 oC before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
attributes.tag: GEO Accession
attributes.value: GSM3753206