title: GSM5823398: CS_seedling_GRO_seq_v2_rep1; Triticum aestivum; OTHER
library_construction_protocol: Leaf tissue of 12-day-old seedlings were collected by flash freezing with liquid nitrogen and followed by nuclei isolation. For GRO-seq, nuclei were isolated by the Percoll gradient procedure as previously described except that the concentration of Triton X-100 was reduced to 0.5%. In vitro run-on, and nascent RNA purification were performed as previously reported20. The obtained nascent RNA was treated with T4 PNK (NEB), followed by TRIzol purification and subject to small RNA library construction using NEXTflex™ Small RNA-Seq Kit v3 (PerkinElmer) according to the manufacturer manual. The cDNA libraries were sequenced on the Illumina NovaSeq platform.For the second time (v2_rep1, v2_rep2, and v2_rep3), to obtain a deeper sequencing coverage at a reasonable sequencing cost, we added a rRNA removal step after nucleus RNA isolation and before the affinity purification of nascent RNA using riboPOOL kit (siTOOLs BIOTECH) . For pNET-seq, nuclei were isolated by a lysis-and-wash procedure as previously described except that the concentration of Triton X-100 was reduced to 0.5%. The RNA Pol II associated RNA were immunoprecipitated using Pol II antibody (Abcam, ab817) and followed by small RNA cDNA library construction. The size selection of nascent RNA was omitted. The cDNA libraries were sequenced on the Illumina NovaSeq platform.