Pure beads

ATAC-seq was performed on 50,000 viable sorted myeloma cells similar to previously described^{43 (link),61 (link),62 (link)}. Briefly, cells were pelleted at 500 × g for 10 min at 4 °C and resuspend in ice-cold nuclei-lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL) and centrifuged at 500 × g for 30 min at 4 °C. Nuclei were resuspended in 22.5 μl of tagmentation DNA buffer (Illumina) with 2 μl tagmentation enzyme (Illumina) at 37 °C for 60 min. Proteins were digested with 2 μg of proteinase K at 40 °C for 60 min. Tagmented DNA was isolated with two rounds of negative (0.6×) and positive (1.2×) size selection with SPRI beads (PureBeads, Kapa Biosystems). ATAC-seq libraries were amplified 12 times with Hifi Polymerase (Kapa Biosystems) and quantitated by qPCR (Kapa Biosystems) and high sensitivity bioanalyzer (Agilent). Sequencing was performed on a NovaSeq 6000 (Illumina) using 150 bp paired-end at the New York Genome Center.

For each strain, three independent biological replicates were collected. Ribosomal RNA was depleted from 5 μg of total RNA using the Ribo-Zero rRNA Removal Kit for Gram-Negative Bacteria (Illumina). The concentrations of the rRNA-depleted samples were determined with the Qubit RNA HS Assay Kit (ThermoFisher). RNA-seq libraries were then generated with the Stranded RNA-Seq Library Preparation Kit for Illumina (KAPA) and NEBNext Multiplex Oligos for Illumina adaptors (NEB) according to the manufacturer’s instructions for 100 ng of starting material and a mean insert size of 200–300 bases. Final libraries were purified with Pure Beads (KAPA). Sequencing library size and integrity were verified with DNA Bioanalyzer analysis (Agilent). Libraries were pooled and sequenced on 2 lanes of 50SE HiSeq. 2500 (Illumina) by the Genomic Services Laboratory at the HudsonAlpha Institute for Biotechnology.

Approximately 100 ng of the concatenated pool was used to prepare PacBio sequencing libraries using the KAPA Hyper Prep Kit. A suitable T-tailed hairpin adapter was first created by self-annealing of oligonucleotide Pr375 (20 μM) using Duplex Buffer and heating for 5 min to 80 °C followed by a slow ramp-down (0.2 °C per second) to 25 °C. Double-stranded DNA concatemers were then subjected to end-repair and A-tailing, and ligated to the hairpin adapters (at roughly a 250:1 ratio of adapters to concatemers) for 30 min at 20 °C. Unreacted T-tailed hairpin adapters and concatenated DNA molecules were removed by adding exonuclease III and exonuclease VII (1 μl of each) to the sample and incubating for 30 min at 37 °C. The resulting library molecules were cleaned-up with KAPA Pure Beads (0.8x ratio), and then quantified using Qubit dsDNA HS Assay. On average the final concentration of the sequencing libraries was between 0.5 and 2 ng/μl.