title: GSM3732476: JR68C_N9ER1N_RNA-Seq; Oryza sativa; RNA-Seq
library_construction_protocol: DNA and total RNA was extracted from snap-frozen tissues using CTAB and TRIzol reagent (Invitrogen), respectively. For bisulfite sequencing, about 1µg of genomic DNA was fragmented by sonication, end repaired and ligated to custom-synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer's (Illumina) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen) as outlined in the manufacturer's instructions. One quarter of the bisulfite-converted libraries was PCR amplified using the following conditions: 2.5 U of ExTaq DNA polymerase (Takara Bio), 5 μl of 10X Extaq reaction buffer, 25 μM dNTPs, 1 μl Primer 1.1, 1 μl Primer 2.1 (50 μl final). PCR reactions were carried out as follows: 95 ̊C 3 min, then 12-14 cycles of 95 ̊C 30 sec, 65 ̊C 30 sec and 72 ̊C 60 sec.  For small RNA libraries, 10 µg of total RNA was loaded on a 15% polyacrylamide, 7M urea gel, and small RNA in the 17-30 nt size range were excised. The 3' miRNA cloning linker 1 (IDT) was ligated to the gel-excised small RNA using truncated T4 RNA ligase 2 (NEB), and ligation products were purified on a 15% polyacrylamide, 7M urea gel. The 5' Illumina RNA linker (5'-rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU-3') was added using T4 RNA ligase (NEB) and ligation products were again purified on a 15% polyacrylamide, 7M urea gel. Purified ligated small RNAs were reverse transcribed using SuperScriptIII (Invitrogen) and PCR-amplified with Phusion High-Fidelity DNA Polymerase (NEB) for 25 cycles (Forward primer: 5'-AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACG-3', Reverse primer: 5'-CAAGCAGAAGACGGCATACGATTGATGGTGCCTACAG-3'). Strand-specific RNA-seq libraries were constructed as described in Wang et al. (2011), beginning with ribosomal RNA-depleted total RNA. TRIzol-extracted total RNA treated with DNase I (Qiagen) was purified using the RNeasy Mini kit (Qiagen) as per manufacturer's instructions. After verifying RNA integrity on a denaturing agarose gel, 5 μg of purified total RNA was depleted for ribosomal RNA using the Ribo-Zero rRNA removal kit for plant seed and root (Epicentre) as per manufacturer's instructions. Ribosomal-depleted total RNA was purified using AM-Pure beads (Beckman Coulter) and fragmented using the Ambion RNA fragmentation kit (Ambion). After overnight precipitation in 75% ethanol, the resuspended pellet was reverse transcribed using Random Hexamer primers (Invitrogen) and SuperScript III reverse transcriptase (Invitrogen). After AM-Pure bead purification. second-strand synthesis proceeded with DNA polymerase I (Invitrogen) and a dUTP mix of 10 mM dA, 10 mM dC, 10 mM dG, and 20 mM dU. After purification of second-strand synthesis products with AM-Pure beads, cDNA was end-repaired using the End Repair Mix LC (Enzymatics) while still attached to AM-Pure beads. Using XP buffer (Wang et al., 2011) for intermediate size selection and washing steps, dA-tailing with Klenow exo minus (NEB) was also performed while cDNA still remained attached to the beads. cDNA was eluted off beads for Y-shape adapter ligation with DNA quick ligase (NEB) and adapters (adapter 1: 5'A*C*ACTCTTTCCCTACACGACGCTCTTCCGAT*C*T 3'; adapter 2: 5'P-G*A*TCGGAAGAGCACACGT*C*T 3'). After purifying adapter-ligated cDNA with AM-Pure beads, dUTP excision was performed with Uracil DNA glycosylase (NEB), and products were directly amplified using Phusion Hot Start 2 DNA polymerase (NEB) and indexed pair-end primers for multiplexing. After dUTP excision and PCR, products were purified and concentrated using AM-Pure beads. Where indicated, duplex-specific nuclease treatment was performed as described by Matvienko et al. (2013). In short, 200 to 300 ng of library product was denatured and hybridized in TMAC buffer (3 M TMAC, 50 mM HEPES, pH 7.5) for 5 hours at 68°C. At the end of 5 hours, duplex-specific nuclease (DSN; Evrogen) was added along with DSN buffer as instructed by the manufacturer. Resulting DSN-treated products were amplified a second time using Phusion Hot Start 2 DNA polymerase (NEB) and indexed pair-end primers for multiplexing. Final DSN-treated library products were gel-purified on 2% agarose to obtain fragments between 300 to 400 bp in length for sequencing. library strategy: Bisulfite sequencing, strand-specific total RNA (with or without duplex-specific nuclease treatment), and small RNA sequencing with Illumina technology. sequencing platform: Illumina HiSeq 2000 (run by the Vincent J. Coates Genomic Sequencing Laboratory at UC Berkeley)
attributes.tag: GEO Accession
attributes.value: GSM3732476