Deep sequencing small RNAs from Gentiana triflora plant affecting kobu-sho disease
Library Preparation - NEBNext Multiplex Small RNA Sample Prep Set for Illumina1 Library Preparation - NEBNext Multiplex Small RNA Sample Prep Set for Illumina1microg total RNA or 10ng small RNA was used for library preparation. 3 'SR Adaptor for Illumina was ligated to the small RNA using 3' Ligation Enzyme. To prevent adaptor-dimer, the excess of 3 'SR Adaptor were hybrid with SR RT Primer for Illumina. 5 'SR Adaptor for Illumina was ligated to the small RNA using 5' Ligation Enzyme and first strand cDNA was synthesized using ProtoScript II Reverse Transcriptase. Each sample was then amplified by PCR using P5 and P7 primers, the PCR product were purified by DNA clean beads. The purified products of 140- 160bp were recovered and cleaned up using PAGE, validated using an Agilent 2100 Bioanalyzer. g total RNA or 10ng small RNA was used for library preparation. 3 'SR Adaptor for Illumina was ligated to the small RNA using 3' Ligation Enzyme. To prevent adaptor-dimer, the excess of 3 'SR Adaptor were hybrid with SR RT Primer for Illumina. 5 'SR Adaptor for Illumina was ligated to the small RNA using 5' Ligation Enzyme and first strand cDNA was synthesized using ProtoScript II Reverse Transcriptase. Each sample was then amplified by PCR using P5 and P7 primers, the PCR product were purified by DNA clean beads. The purified products of 140- 160bp were recovered and cleaned up using PAGE, validated using an Agilent 2100 Bioanalyzer.