Determination of eRNA 3' Ends

To determine the 3′ end of the different eRNA species transcribed from the CNS2 region, we used a previously described RNA circularization method (Couttet et al., 1997 (link); Kim et al., 2010 (link)). Briefly, 5μg of DNase treated RNA were incubated with 0.5 units of tobacco acid pyrophosphatase (Thermo Fisher) at 37°C for 1 h to expose the phosphates at the 5′ ends of all RNAs. Decapped RNA was purified and circularized with 5 units of T4 RNA ligase, which catalyzed phosphodiester bond formation between the generated 5′ phosphate and the 3′ hydroxyl group. cDNA synthesis from circularized RNA was performed with SuperScript III and random hexamers with the following program: 25°C for 5 min, 50°C for 1 h and 70°C for 15 min. After reverse transcription, 1μl of cDNA was used to amplify 5′-3′ junctions with primer pairs specifically design to that end (Supplementary Figure 1A). For sequencing, PCR products were cloned with TOPO-TA cloning kit (Thermo Fisher, K4575J10) according to manufacturer’s instructions.