title: GSM2228823: MT_bio2_sRNA; Citrus sinensis; ncRNA-Seq
library_construction_protocol: Approximate 2 to 3 g of powdered material was subjected to each RNA extraction, including 10 mL buffer extraction, 5 mL chloroform-isoamylalcohol extraction (twice), 5 mL isopropanol precipitation, 5 mL 75% ethanol cleaning and RNA purification as described by Camacho-Villasana YM et al. For Small RNA library, the small RNAs (~18–30 nt) were purified from 10 μg of total RNA by polyacrylamide gel electrophoresis, and ligated first to a 5’ RNA adaptor and then to a 3’ RNA adaptor. A reverse transcription reaction was followed by several cycles of PCR to obtain sufficient product for Sequencing by Synthesis (SBS) sequencing via Solexa technology. For degradome library, poly(A+) RNA molecules were isolated from 200 μg of total RNA using the Oligotex mRNA kit (Qiagen), and then a 5’ RNA oligonucleotide adaptor containing an MmeI recognition site was ligated to the 5’-phosphate of the poly(A+) RNA by T4 RNA ligase. This was followed by purification of the ligated products using the Oligotex kit. Subsequently, five PCR cycles were performed on the products of a reverse transcription reaction which were then digested with MmeI and ligated to a 3’ double DNA adaptor. Finally, the ligation products were amplified with 20 PCR cycles, gel-purified, and subjected to SBS  sequencing by the Illumina sequencer.
attributes.tag: GEO Accession
attributes.value: GSM2228823