GSM5381368: HEK_siDBR1_B; Homo sapiens; RNA-Seq
To enrich for lariats, poly(A)- and rRNA depleted RNA was prepared as follows. Total RNA was purified from HEK293 cells (10 cm plates) using the Direct-zol RNA Miniprep Kit (Zymo Research) and poly(A)+ RNA was depleted using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) following the manufacturer's instructions with some modifications. Briefly, unbound RNA (supernatant) was further purified using the RNA Clean & Concentrator Kit (Zymo Research). Poly(A)+ RNA depletion was performed twice followed by rRNA depletion using NEBNext® rRNA Depletion Kit (NEB E6310). RNA libraries were constructed using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB, E7760) with the following modification. Reverse transcription was performed using Superscript IV at 57 oC for 30 minutes that showed higher efficiency to reverse transcribe branch points FigureS14). Similar library construction approach was followed to detect lariats in nascent RNA obtained by 4SU pulse chase. 150 bp paired end sequencing was performed on the Ilumina NextSeq 500 or HiSeq X Ten platforms