title: GSM3619553: WT1, WT1XOC; Oryza sativa; ncRNA-Seq
library_construction_protocol: Infiltrated leaf part was collected 24 h after inoculation, snap frozen in liquid nitrogen, and stored at −80°C immediately. Bacteria cultures were collected by centrifugation and the medium discarded. Snap frozen the cell pellets in liquid nitrogen, then stored at −80°C immediately. Before cDNA library preparation, ribosomal RNA was removed using the Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA), and the rRNA-free residue was cleaned by ethanol precipitation. Subsequently, sequencing libraries were generated using the rRNA-depleted RNA with the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations. Briefly fragmentation was performed using divalent cations under elevated temperatures in NEB Next First Strand Synthesis Reaction Buffer. First-strand cDNA was synthesized using random hexamer primers and the M-MuLV Reverse Transcriptase (RNaseH-). Second-strand cDNA synthesis was performed subsequently using DNA Polymerase I and RNase H. In the reaction buffer dNTPs, dTTP was replaced with dUTP. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activity. After adenylation of the DNA fragment 3' ends, the NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization.   To preferentially select cDNA fragments 150-200 bp in length, the library fragments were purified with the AM Pure XP system (Beckman Coulter, Beverly, MA, USA). After that, 3 μL of the USER Enzyme (NEB, USA) was incubated with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by incubation for 5 min at 95°C prior to the PCR. The PCR was performed with the Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.   Finally, the products were purified (AM Pure XP system), and the library quality was assessed on the Agilent Bioanalyzer 2100 system.
attributes.tag: GEO Accession
attributes.value: GSM3619553