title: GSM8975359: K562 chromatin SPLASH, biological replicate 2; Homo sapiens; OTHER
library_construction_protocol: Chromatin-enriched RNA was extracted with TRIzol and further purified on Qiagen RNeasy Mini columns. 20 ug of chromatin-enriched RNA were treated with Turbo DNase (Invitrogen, 1x DNase buffer, 0.1U/μL Turbo DNase, 0.4U/ul SUPERase·In) at 37°C for 30 minutes to remove genomic DNA, followed by ribosomal-RNA (rRNA) depletion using RNase H–based methods. Briefly, 20 μg of home-made ribosomal RNA-specific single stranded DNA probes were incubated with ~20ug of gDNA-removed chromatin RNA in probing buffer (100mM Tris-HCl PH7.0, 200mM NaCl) at 95°C for 5 minutes, followed by gradual temperature decrease at 0.1°C /s ramp rate to 22°C. The annealed DNA-RNA hybrid was digested with 0.5U/μL RNaseH (NEB) in 1x RNaseH reaction buffer at 37°C for 1 hour, followed by DNA oligo removal with 0.1U/ μL Turbo DNaseI at 37°C for 1 hour. The rRNA depleted RNA was then biotinylated via a copper-free click reaction (2 mM Click-iT Biotin PEG4 alkyne, 25 °C, 1 hour) and cleaned up with RNA Clean & Concentrator-5 (Zymo). 1μg of biotin-labelled RNA was diluted to 50 ng/ μL using 20 μL of nuclease free water, mixed with 20 μL of 2× fragmentation buffer( 9 mM MgCl2, 225 mM KCl, 150 mM Tris-HCl pH 8.3), incubated at 95 °C for 5 minutes, and immediately cooled on ice for 2 minutes. 2 μL of 0.5 M EDTA was added to chelate Mg²⁺, and the fragmented RNA was used directly without purification for streptavidin pull-down and proximity ligation as described previously (Aw et al 2016) . Notably, after proximity ligation, 3ʹ adaptors were ligated to the RNA on-beads with T4 RNA ligase 2 truncated KQ (NEB) in 30 μL of ligation mix (1× RNA ligase buffer, 20 U/μL T4 RNA ligase 2 TnKQ, 33 ng/μL 3ʹ adaptor, 0.67 U/μL SUPERase·In, 15 % PEG-5000) at 25°C for 2.5 hours, followed by RNA elution and clean-up as previously described (Aw et al 2016). Eluted RNA was reverse-cross-linked by 254-nm UV irradiation for 5 minutes on ice. Reverse transcription was performed with SuperScript IV at 50 °C for 1 hour using 1 μM 3ʹ adaptor–specific primer (5' -TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3') and 1 μM template-switch oligo (TSO) containing a 10-nt unique molecular identifier (UMI) (5' - CTT TCC CTA CAC GAC GCT CTT CCG ATCT NNNNNNNNNN TAC rGrGrG /3phos/ -3'). cDNA fragments of 150–300nt were size-selected on a 6 % urea-PAGE gel, then amplified with NEBNext Multiplex Oligos using Q5 DNA polymerase (NEB) for 12 cycles. PCR products were purified with 0.9× Agencourt AMPure XP beads, quantified with Qubit High-Sensitivity DNA kit, and submitted for high-throughput sequencing using Novaseq-X-plus machine (Illumina).