Pippin prep 1.5 agarose cassette

A multiplexed amplicon library covering the 16S rDNA gene V4 region was generated from human stool sample DNA. Bacterial genomic DNA was extracted using the Mo Bio Power Fecal DNA Isolation kit (Mo Bio Laboratories, Carlsbad, CA) according to the manufacturer’s instructions for high yields of DNA with the following modifications to increase yields from difficult to lyse microbes. An additional bead beater step using the Faster Prep FP120 (Thermo) at 6 meters/second for 1 min was used instead of vortex agitation. Incubation with buffers C2 and C3 was increased to 10 min at 4°C.
Starting nucleic acid concentrations were determined by a Qubit Fluromoter (Life Technologies, Carlsbad, CA). A multiplexed amplicon library covering the 16S rDNA gene V4 region was prepared using the protocol of with dual-index barcodes.^{17 (link)} The aggregated library pool was size selected from 300–500 bp on a pippin prep 1.5% agarose cassette (Sage Science, Beverly, MA) according to the manufacturer’s instructions. The concentration of the pool was measured by qPCR (Kapa Biosystems, Willmington, MA) and loaded onto the MiSeq Illumina instrument (300 bp kit) at 6–9pM with 40% phiX spike-in to compensate for low base diversity according to Illumina’s standard loading protocol.

Bone marrow-derived MNC cells were isolated as described above prior to a 6 hour treatment with DMSO vehicle or A-485 (1μM). MM cells were then specifically isolated using the EasySep™ Human Whole Blood and Bone Marrow CD138 Positive Selection Kit II (Stemcell Technologies) and collected for 3’-mRNA-seq and ChIP-seq. 0.5–1e5 CD138⁺ MM cells were then crosslinked, lysed, sonicated, and immunoprecipitation performed (using H3K27ac antibody, ab4729, Abcam), following the manufacturers instructions for the LowCell# ChIP kit (Diagenode, Belguim). Next, immunoprecipitated DNA was isolated using the ChIP DNA Clean and Concentrator Kit (Zymo Research, D5205) and quantified using the Qubit dsDNA HS (ThermoFisher Scientific). Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Ilumina (New England Biolabs, E7645) according to the manufacturers instructions. Resultant libraries were size selected for fragments between 200–500 b.p. using a Pippin Prep 1.5% agarose cassette (Sage Science, Beverly, USA) and sequenced on the Illumina NextSeq 500 to obtain 75 b.p. single-end reads.