title: GSM7138251: ChrRNA-seq_APA_rep2; Homo sapiens; OTHER
library_construction_protocol: HeLa cells were transfected in 15-cm plates as above with gene-specific gRNAs alone or a mix of gene-specific gRNAs for APA experiments. With three quarters of transfected cells, chromatin RNA was extracted as described in (Sousa-Luís et al 2021 Mol Cel) with the following modifications: chromatin pellet was digested with 2 µl of Turbo DNase (Life Technologies) in 200 µl of High Salt Buffer (10 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 10 mM MgCl2) for 15 min at 37C, then treated with Proteinase K in 0.2% SDS for 10 min at 37C. Chromatin RNA was extracted by phenol/chloroform and a second round of Turbo DNase digestion was performed, followed by RNA extraction using Trizol. ne microgram of chromatin RNA was ribodepleted using RiboCop rRNA Depletion kit (Lexogen), followed by library preparation using NEBNExt Ultra II directional RNA kit.