title: NextSeq 500 sequencing; HyPro-seq analysis of HeLa and ARPE-19 cells
library_construction_protocol: HeLa or ARPE-19 cells grown in 10 or 15 cm dishes, respectively, were fixed with 0.5 mg/ml dithiobis(succinimidyl propionate) (DSP; Thermo Fisher Scientific, cat# 22585) in 1×PBS for 30 min at room temperature. Cells were then washed three times with 1×PBS and 20 mM Tris-HCl, pH 8.0, 5 min each wash, permeabilized with 70% ethanol at room temperature for 1 hour, equilibrated in 2×SSC and 10% formamide (Thermo Fisher Scientific) for 5 min, and hybridized with digoxigenin-labeled oligo probes in 2×SSC, 10% formamide and 10% dextran sulfate (Sigma Aldrich, cat# D8906) overnight at 37°C. To achieve comparable labeling intensities, we adjusted probe concentration according to the RNA \"bait\" expression level: 45S-specific probe set, 5 nM; NEAT1-specific probe set, 25 nM; probe targeting (UC)n repeats of PNCTR, 50 nM. Following the hybridization, samples were washed with 2×SSC and 10% formamide at 37°C for 30 min and 1×SSC at room temperature for 15 min and blocked with 0.8% BSA in 4×SSC (HyPro blocking buffer) and 100 units/ml murine RNase inhibitor (New England Biolabs, cat# M0314) at room temperature for 30 min. We then incubated the samples with 2.7 μg/ml HyPro enzyme in HyPro blocking buffer at room temperature for 1 hour. Unbound HyPro was washed off with 4×SSC, 4×SSC and 0.1% Triton-X 100, 4×SSC for 10 min each, and left in 1×PBS for 5 min. In HyPro infusion controls, the cells were bathed in 1×PBS containing 5.4 μg/ml of HyPro enzyme for 5 min. Proximity biotinylation was then carried out by the addition of an equal volume of 1×PBS containing 1 mM biotin-phenol (Caltag Medsystems, cat# CDX-B0270) and 0.2 mM hydrogen peroxide (Sigma Aldrich, cat# H1009) and gently agitating the plates for 1 min. The reaction was quenched by quickly rinsing the plates three times with 5 mM TROLOX (Sigma Aldrich, cat# 238813) and 10 mM sodium ascorbate (Sigma Aldrich, cat# A4034) in 1×PBS.  To extract RNA from HyPro-labeled samples, cells were lysed in high-SDS RIPA buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% sodium deoxycholate and 0.5% SDS) supplemented with 10 mM sodium ascorbate, 5 mM Trolox, 50 mM DTT and 100 units/ml of murine RNase inhibitor. Cells were incubated in 1 ml/10-cm or 2 ml/15-cm dish of this buffer on ice for 10 min, scraped off the plates and further incubated on ice for 10 min. The samples were then sonicated using a Bioruptor system (Diagenode) equipped with a refrigerated ice bath, for 5-10 cycles of 30 sec ON / 30 sec OFF at the HIGH power setting. The lysates were then incubated at 37°C for 30 min to reverse crosslinks, supplemented with 400 μg of proteinase K (Thermo Fisher) and incubated at 50°C for 1 hour. Three volumes of Trizol LS (Thermo Fisher) was then added to the lysate, the phases were separated by chloroform, and total RNA was extracted from the aqueous phase using Purelink RNA miniprep kit (Thermo Fisher), as recommended. Small aliquots were set aside at this point for RT-PCR quality control and total RNA sequencing (the latter was done for HeLa HyPro infusion controls only; samples HeLa_CTRL_INPUT_...). The rest of RNAs was processed further to isolate biotinylated species. To capture biotinylated RNAs, we used 10 μl of MyOne streptavidin C1 magnetic beads (Thermo Fisher Scientific, cat# 11205D) per 20 μg of total RNA. The beads were washed three times with the B&W buffer (5 mM Tris-HCl, pH 7.5, 5 mM EDTA, pH 8.0, 1 M NaCl, 0.1% Tween-20), once in solution A (100 mM NaOH and 50 mM NaCl), once in solution A with 0.1% Tween-20, once in 100 mM NaCl, and once in 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 0.2% Tween-20. The beads were then resuspended in 150 µl of 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 0.2% Tween-20 and incubated with an equal volume of RNA eluted in nuclease free-water (Thermo Fisher Scientific, cat# AM9939) for 2 hours at 4°C with continuous rotation. The beads were collected using a DynaMag™-2 Magnet (Thermo Fisher), washed three times with B&W buffer and resuspended in 100 µl of 100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS and 0.80 µg/µl proteinase K. The beads were then incubated at 50°C for 45 min in a thermomixer and mixed with 300 µl of Trizol LS and 80 µl of chloroform. Biotinylated RNAs were then purified from the aqueous phase using an RNA Clean & Concentrator kit (Zymo Research). RNA samples were depleted of mature ribosomal RNAs using a NEBNext® rRNA Depletion Kit (New England Biolabs,cat# E6350), and used to prepare stranded sequencing libraries with a NEBNext Ultra II Directional RNA library Prep kit for Illumina and barcoded primers (New England Biolabs, cat# E7765), as recommended. The barcoded libraries were quantified using NGSBIO Library Quant Kit Blue for Illumina® (PCR Biosystems, cat# PB71.15-01) and pooled prior to sequencing.