GSM5593713: FLEP_seq_fpa; Arabidopsis thaliana; OTHER
Two grams of seedlings were ground with liquid nitrogen into fine powder and transferred to an ice-cold 50 ml tube with 10 ml Honda buffer (0.44 M Sucrose, 1.25% (wt/vol) Ficoll, 2.5% (wt/vol) Dxtran T40, 20 mM HEPES-KOH pH 7.4, 10 mM MgCl2, 0.5% (wt/vol) Triton X-100, 1 mM DTT, 1× protease inhibitor (Roche), 100 ng/μl tRNA). After homogenization by vortexing, the homogenate was filtered through one layer of miracloth. Another 10 ml Honda buffer was added to the filter to wash the remaining plant material. After centrifugation at 4 ℃ and 3,500 g for 5 min, the supernatant was collected as the cytoplasmic fraction and the pellet was resuspended and washed once with another 20 ml Honda buffer. Then the pellet was resuspended in 1 ml Honda buffer (0.44 M Sucrose, 1.25% (wt/vol) Ficoll, 2.5% (wt/vol) Dxtran T40, 20 mM HEPES-KOH pH 7.4, 10 mM MgCl2, 0.5% Triton X-100, 1 mM DTT, 500 ng/μl tRNA, 2× proteinase inhibitor (Roche), 10 μl RNase inhibitor (RNase out, Thermo)) and centrifuged at 4 ℃, 8000 g for 1 min. The supernatant was completely removed and the nuclei pellet was retained as the nuclear RNA fraction. After rRNA removal by using RiboMinus Plant Kit for RNA-Seq (Invitrogen, A10838-08), the nuclear RNA was mixed with 50 pmol 3' adapter (5'-rAppCTGTAGGCACCATCAAT–NH2 -3', NEB 1315) at 65 ℃ for 5min, and then placed on ice for more than 1 min. 2 μl 10× T4 RNA ligase reaction buffer (NEB, M0242), 10 μl 50% PEG 8000 (NEB, M0242), 1 μl 40 U μl-1 RNaseOUT (Invitrogen, 10777019) and 1 μl T4 RNA ligase 2, truncated K227Q (NEB, M0242) were added to the RNA. The ligation reaction was performed at 16 ℃ for 10 h. The ZYMO RNA Clean & Concentrator kit (ZYMO, R1013) was used for RNA purification. Reverse transcription and template-switching were performed using the SMARTer PCR cDNA Synthesis Kit (Takara, 634926) with minor modification. The SMART CDS Primer II A was replaced by the custom primer (5'-AAGCAGTGGTATCAACGCAGAGTACATTGATGGTGCCTACAG-3') which is complementary to the 3' adapter sequence. To minimize the PCR bias resulting from over-amplification, PCR cycle optimization was performed to determine the best cycle number for cDNA amplification. VAHTS DNA Clean Beads (Vazyme, N411) was used to purify PCR products. 200 fmol cDNA was used to construct the Nanopore library (using Ligation Sequencing Kit, SQK-LSK109, Oxford Nanopore Technologies). Libraries were loaded onto R9.4 flow cell (Oxford Nanopore Technologies) and sequenced on MinION device for ~48 hr. FLEP-seq