title: GSM5507850: GROseq Mock_for_DC3000_rep2; Arabidopsis thaliana; OTHER
library_construction_protocol: GRO-seq libraries were constructed as previously described with minor modifications (Wang et al, 2011). Briefly, nuclei were resuspended by nuclear run-on buffer and were incubated for 5 min at 30°C in water bath, followed by proteinase K digestion and acidic phenol/ chloroform purification. After fragmentation, nascent RNAs with 5'-bromo-UTP were immuno-precipitated twice by using beads conjugated with an anti-BrdU antibody (Santa Cruz, sc-32323 AC). After poly-A tail addition and reverse transcription, the resulting first-strand cDNA was separated in a 10% TBE-urea polyacrylamide gel. cDNA that is 100–400 nt long was gel-recovered and self-ligated using CircLigase (Epicentre). The circularized single-stranded cDNA was relinearized with APE 1 (NEB) and then PCR-amplified using Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific) at optimal cycle numbers. The PCR products were separated in a 10% native TBE polyacrylamide gel and the 140–225 bp products were recovered. All libraries were sequenced on Illumina NovaSeq 6000 platform (SE 75 bp reads). Total RNA was extracted using RNeazy Plant Mini Kit (QIAGEN, 74904). The RNA sequencing (RNA-seq) libraries were generated using NEXTFLEX ®Rapid Directional qRNA-SeqTM Kit (Bioo Scientific, NOVA-5130-01D) according to the manufacturer's instructions.