alias: GSM2888871
title: GSM2888871: ΔCYC2 x ΔCYC2; Tetrahymena thermophila; RNA-Seq
library_construction_protocol: Samples were collected from 5ml of conjugational Tetrahymena WT and mutant cells (density: 2.5 x 105 cells/mL) and transferred into 2 ml RNase-free tube. 1ml RNAiso was added into the tubes to lyse cells. The tube placed 4 ℃ centrifuge, 12800 rpm for 1 minute，Absorb supernatant to a spin column，4℃，8800 rpm for 20 seconds. Absorb supernatant in collection tube to a 1.5ml RNase-free tube，add 0.5 volumes of ethanol, followed by all of the liquid was transferred to a spin column, 4℃，8800 rpm for 20 seconds. Now RNA adsorbed on a silicon membrane inside the spin column. A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library 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（RNase H-）. 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 37°C for 15 min followed by 5 min at 95 °C 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.
identifiers.namespace: GEO
identifiers.id: GSM2888871