Pippinht instrument

NGS libraries from DRIP DNA and inputs were constructed according to the manufacturer’s instructions (KAPA Hyper Prep Kit: Roche, cat. no. #KK8502 kit and single-indexed adapters from Illumina, cat. no. #IP-202–1012 + IP-202–1024. Dual-indexed libraries were constructed with Roche, cat. no. #KK8722). Library fragment sizes were restricted to between 100–500 bp with a PippinHT instrument (Sage Science).
NGS libraries from total, purified RNA were constructed with the SMARTer Stranded RNA-seq Kit (Takara, cat. no. #634839) according to the manufacturer’s instructions for first-strand cDNA synthesis, purification of first-strand cDNA using SPRI beads, RNA-seq library amplification via PCR (with 12 PCR cycles for 10 ng of starting RNA), and SPRI cleanup.
All library sizes and concentrations used for molarity calculations were confirmed on an Agilent 2100 Bioanalyzer and a Qubit 4 fluorometer (ThermoFisher Scientific) with a Qubit dsDNA HS Assay kit (ThermoFisher Scientific, cat. no. #Q32854), respectively.
DRIP- and RNA-sequencing was performed on an Illumina HiSeq 2500 (single-indexed libraries). All sequencing was performed at the New York Genome Center, New York, NY, 2×150bp, to a minimum read depth of 50,000,000 reads. See Supplementary Table 2 for information about additional sequencing metrics.

PacBio HiFi data were generated from the HG00733 lymphoblastoid cell line as previously described (Logsdon et al. 2021 (link)) with modifications. Briefly, DNA was extracted from 4.3 × 10⁶ cells using the Monarch HMW DNA Extraction Kit for Cells and Blood (New England Biolabs) with 1400 rpm lysis speed. After UV absorption and fluorometric quantification (Qubit High Sensitivity DNA kit, Thermo Fisher Scientific) on the DS-11 FX instrument (Denovix) and evaluation of DNA integrity on FEMTO Pulse (Agilent), 12 μg of DNA was prepared for sequencing using Megaruptor 3 shearing (Diagenode, settings 19/31) and the Express Template Prep Kit v2 and SMRTbell Cleanup Kit v2 (PacBio). The library was size-selected on a PippinHT instrument (Sage Science) using a 15 kbp high-pass cut. Five SMRT Cell 8Ms were run on a Sequel II instrument using Sequel II chemistry C2.0/P2.2 with 30-h movie times, 2-h pre-extension, and adaptive loading targets of 0.8–0.85 (PacBio). Circular consensus calling was performed with CCS version 6.0.0 (SMRT Link v.10.1) and reads with estimated quality scores ≥Q20 were selected for downstream analysis.

Small RNA sequencing libraries were constructed using a modified Illumina small RNA sequencing library construction method [38 (link)]. Briefly, T4 RNA ligase enzymes were used to attach Illumina compatible adapter sequences to the 3’ and 5’ ends of the RNA. These adapters each contain four degenerate bases at the RNA-adapter ligation site. The adapters were purchased from Integrated DNA Technologies (Coralville, IA, USA). Following adapter ligation, the libraries were incubated with a single-stranded binding protein (Promega, Madison, WI), 5’-deadenylase (New England Biolabs, Ipswich, MA, USA), and RecJf exonuclease (NEB) to reduce adapter-dimer formation. Then, cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, Waltham, MA, USA) and the small RNA library was amplified by PCR (polymerase chain reaction) with a high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA), an Illumina universal primer, and selected indexed-primers. PCR products were cleaned with Ampure XP beads (Beckman Coulter, Indianapolis, IN, USA) and the library was size-selected using a PippinHT instrument (Sage Science, Beverly, MA, USA). The target range was set at 134–162 nt to recover inserts 20–22 nt in length.