title: GSM4512913: rdr6_prophase_I_degradome_rep3; Oryza sativa; RNA-Seq
library_construction_protocol: Libraries for degradome sequencing were constructed based on the PARE method  with considerable modifications that allow use of low-input RNAs. In detail, 5' RNA adaptor was first heated at 75 °C for 3 min and then immediately chilled on ice for 2 min. For 5' RNA adapter ligation, total RNA (3 μL) was added to a solution composed of 1 μL of T4 RNA ligase buffer, 1 μL of 10 mM ATP, 0.5 μL of RNasin, 0.5 μL of 10 μM treated RNA adaptor, 3 μL of PEG8000 (50% v/v), and 1 μL of T4 RNA ligase 1 and incubated at room temperature for 1 h. Then 1 μL of 10 mM dNTP and 0.5 μL of 10 μM reverse transcription primer Oligo(dT)VN  were added. After incubating at 75°C for 5 min and chilling on ice for 2 min, 5 μL of first strand RT buffer, 5 μL of 5 M betaine (Sigma, 61962), 1.25 μL of 0.1 M DTT, 0.9 μL of 1/6 M MgCl2, 0.5 μL of Ribonuclease inhibitor (Promega, N2511), and 1 μL of SuperScriptIII reverse transcriptase (Invitrogen, 18080-085) were added for reverse transcription. The condition for reverse transcription was 44°C for 1 h followed by 15 cycle of 50°C for 2 min and 44°C for 2 min. After reverse transcription, the products were purified with 2 × AMPure XP beads (v/v). Then the products (20 μL) were pre-amplified using GXL DNA polymerase (Takara, R050A) with 5' biotin-labelled forward primer F1 and reverse primer R1 in a solution composed of 10 μL of 5 × GXL buffer, 4 μL of 2.5 mM dNTP, 1.5 μL of 10 μM F-primer, 1.5 μL of 10 μM R-primer, 2 μL of GXL DNA polymerase, 11 μL of ddH2O. The PCR was performed at 98°C for 2 min followed by 20 cycle of 98°C for 10 s, 60°C for 15 s, 68°C for 3 min and then 68°C for 5 min. After PCR, 1 μL of Exonuclease I (NEB, M0293L) and 5.7 μL of 10 × Exonuclease I buffer were added to the sample and the mixture was incubated at 37°C for 1 h. Then 19 μL of 4 × binding buffer (40 mM Tris-HCl, pH 8.0, 2 mM EDTA, 4 M NaCl) was added and the mixture was incubated with DynabeadsMyOne Streptavidin C1 beads (Invitrogen, 65001) at room temperature for 30 min with rotation. The beads were washed once with 1 × TWB buffer (10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20 v/v) and 3 times with 1 × EBT buffer (10 mM Tris-HCl, pH 8.0, 0.02% Triton X-100 v/v) and then digested with MmeI (NEB, R0637L) in a solution composed of 3 μL of 10 × NEB buffer 2, 3 μL of 0.5 mM SAM, 2 μL of Mme I and 22 μL of ddH2O at 37°C for 2 h with rotation. The beads were washed once with 1 × TWB buffer and 3 times with 1 × EBT buffer. The DNA products bound to the beads were ligated to 3' DNA adaptor in a solution composed of 3 μL of 2 × quick ligase reaction buffer, 1 μL of 10 mM adaptor, 1 μL of quick ligase (NEB, M2200L) and 25 μL of ddH2O at room temperature for 30 min with rotation. The 3' DNA adapter was prepared by annealing of two DNA oligos in 1 × annealing buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 50 mM NaCl). The beads were washed once with 1 × TWB buffer and 3 times with 1 × EBT buffer and resuspended in 30 μL of 0.05% Triton X-100. The samples were incubated at 80°C for 30 min with rotation to elute the DNA products. Then the DNA products were subjected to final PCR amplification using GXL DNA polymerase and primers F2 and R2. PCR was performed at 98°C for 2 min followed by 22 cycle of 98°C for 10 s, 60°C for 15 s, 68°C for 20 s and then 68°Cfor 5 min. The final products were resolved on a PAGE gel and ~140-bp bands were excised and purified for sequencing on an Illumina HiSeqX-Ten platform. Total RNA was extracted using TRIzol (Takara, 9109). MgCl2 (final concentration: 0.2 M) was added to prevent loss of sRNAs with low GC content. The extracted RNA was stored at -80°Cin LoBind tubes (Eppendorf, 022431021).
attributes.tag: GEO Accession
attributes.value: GSM4512913