GSM5940376: Dark2; Arabidopsis thaliana; RNA-Seq
Total RNA extraction of seedlings was carried out using RNeasy Plant Mini Kit (Qiagen) following manufacturer's instructions The 3'READS+ procedure has been previously described (Zheng et al., 2016). Briefly, polyA+ RNA in 1 µg of total-cell RNA was captured using 10 µl of oligo(dT)25 magnetic beads (NEB) in 100 µl of 1x Binding Buffer (10 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20) and fragmented on the beads using 1.5 U of RNase III (NEB) in 30 µl of RNase III Buffer (10 mM Tris-Cl pH 8.3, 60 mM NaCl, 10 mM MgCl2, and 1 mM DTT) for 15 min at 37 °C. After washing away unbound RNA fragments using Binding Buffer, polyA+ fragments were eluted from the beads using TE Buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5) and precipitated using ethanol, followed by ligation to 3 pmol of heat-denatured 5'-adapter (5'-CCUUGGCACCCGAGAAUUCCANNNN-3') in the presence of 1 mM ATP, 0.1 µl of SuperaseIn (ThermoFisher Scientific), and 0.25 µl of T4 RNA ligase 1 in a 5 µl reaction at 22 °C for 1 hr. Ligation products were captured using 10 pmol of biotin-T15-(+TT)5 attached to 12 µl of Dynabeads MyOne Streptavidin C1 (ThermoFisher Scientific). After washing with Washing Buffer (10 mM Tris-Cl pH 7.5, 1 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20), RNA fragments on the beads were incubated with 0.01 U/µl of RNase H (Epicentre) for 30 min at 37 °C in 30 µl of RNase H buffer (50 mM Tris-Cl pH 7.5, 5 mM NaCl, 10 mM MgCl2, and 10 mM DTT). After washing with RNase H Buffer, RNA fragments were eluted from the beads in Elution Buffer (1 mM NaCl, 1 mM EDTA pH7.5, and 0.05% TWEEN 20) at 50 °C, precipitated using ethanol, and then ligated to 3 pmol of heat-denatured 5'-adenylated 3'-adapter (5'-rApp/NNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC-3') using 0.25 µl of T4 RNA ligase 2 (truncated KQ version) for 1 hr at 22 °C in a 5 µl reaction containing 15% PEG 8000 and 0.2 µl of SuperaseIn. Ligation products were precipitated and reverse-transcribed using M-MLV reverse transcriptase, and PCR-amplified using Phusion high-fidelity DNA polymerase (NEB) and bar-coded PCR primers (Sigma-Aldrich) for 15 cycles.