High sensitivity bioanalyzer

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.

A modified version of Cel-seq2 was used to generate single cell cDNA libraries. Briefly, the sorted 384-well plates were defrosted on ice and the cells were lysed and the primers annealed at 65 °C. Primers contained a 24 bp polyT stretch, a 6 bp unique molecular identifier (UMI), a cell-specific 8 bp barcode, the 5′ Illumina adaptor and a T7 promoter. Reverse transcription, second strand synthesis and in vitro transcription were performed according to the Cel-seq2^{68 (link)} with the exception of the cDNA cleanup step prior to IVT, whereby the volume of the Agencourt AMPure XP beads (A63880, Beckman Coulter) was 5% of the total volume of beads used in the original protocol. Following IVT, the aRNA was fragmented, cleaned up and reverse transcribed with a random hexamer primer containing the sequence complimentary to the 3’ Illumina adaptor. Fragments containing both adaptors were selected for by PCR using TruSeq Small RNA primers (Illumina) and cleaned up using a 0.7X bead to cDNA ratio. The quality and concentration of the final cDNA library was checked using a High Sensitivity Bioanalyzer (Agilent). Libraries were pooled at equimolar ratios and sequenced on the Illumina Nextseq.

The genomic DNA of bulk cells (1 × 10⁶ cells) was extracted using the ALLPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany, Cat. 80204) according to the manufacturer’s manual. Quantified 50 ng genomic DNA was used to prepare the paired-end library using the TruePrep DNA library Prep Kit V2 for Illumina (Vazyme, Cat. TD-501). The quality and concentration of DNA fragments in the DNA libraries generated were assessed using High-Sensitivity Bioanalyzer (Agilent, Santa Clara, CA, USA). The prepared library was then subjected to Illumina HiSeqXten Sequencer (San Diego, CA, USA) with the paired-end 150 bp read option.