Chromatin isolation by RNA purification

For affinity capture of complexes containing lncRNA033862 and chromatin, we designed 10 tiled antisense probes covering the sequence of XLOC033862 using an online design tool (www.singlemoleculefish.com). Probes were numbered according to their position and separated into two pools containing either odd (1, 3, 5, 7 and 9) or even-numbered probes (2, 4, 6, 8 and 10; for sequences see Supplementary Table 2).

ChIRP was performed according to a previously published protocol.⁵⁸ Briefly, approximately 25–30 million cells were harvested and cross-linked with 1% glutaraldehyde (Sigma-Aldrich) for 10 min prior followed by quenching with 1.25 M glycine for 5 min. Cell pellets were collected by centrifugation and lysed in a buffer containing 50 mM Tris, 10 mM EDTA, 1% SDS, 1 mM PMSF (Sigma-Aldrich), 1x Protease inhibitor (Roche) and 0.1 U/μl RNAse inhibitor (Life Technology). Lysates were sonicated to shear the DNA to lengths of 100–500 bp using an Ultrasonic Broken Instrument (Branson, Shanghai, China) in a 4 °C water bath at a setting of 10 cycles for 20 s, interspersed with a rest period of 30 s. Sonicated samples were continuously centrifuged at 16 100 g for 10 min at 4 °C. Supernatants were hybridized separately with each of the two pools of 3'-biotinylated probes (GenScript, Suzhou, China; 1 μl of 100 pmol/μl probes per 1 ml chromatin) in a buffer consisting of 500 mM NaCl, 1%SDS, 100 mM Tris 7.0, 10 mM EDTA, 15% formamide, 1mM PMSF, 1x protease inhibitor and RNAse inhibitor at 37 °C for 4 h with gently shaking. Following hybridization, samples were incubated with Dynabeads MyOne Streptavidin C1 (Life Technology) at 37 °C for 30 min. After five washes in 2x SSC (Life Technology) supplemented with 0.5% SDS and 1mM PMSF, beads with conjugated RNP–chromatin complexes were separated using a DynaMag-2 magnetic strip (Life Technology).