Ercc rna spike in control mix

The RNA was isolated from TRIzol samples using Direct-zol RNA MiniPrep Plus (Zymo) following the manufacturer’s protocol and subsequently treated with TURBO DNase (ThermoFisher). The RNA was purified using RNA Clean & Concentrator Kit (Zymo), and to normalize for differences in library size, ERCC RNA Spike-In Control Mix (ThermoFisher) was added into each sample following the manufacturer’s protocol. The integrity of the samples was analyzed using Fragment Analyzer Service provided by QB3 at UC Berkeley. The samples were sent to QB3 for ribodepletion and library generation. After multiplexing the samples, they were sequenced on the HiSeq 4000 (for dox-induction experiment) or NovaSeq 6000 (for VACV infection experiment) to obtain paired-end 150 nucleotide reads.

Total RNAs were extracted by the RNeasy mini kit (QIAGEN). Genomic DNA was removed on-column by RNA-free DNase I for 15 min at room temperature. Total RNA amounts were determined by NANODROP 2000/2000c (Thermo Fisher Scientific) and assessed for quality using the TapeStation (Agilent, #5067-5576). Samples were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, #E7760L), NEBNext rRNA Depletion Dit (Human/Mouse/Rat) (NEB, #E6310X), NEBNext Multiplex Oligos for Illumina (NEB, #E6440L) and ERCC RNA Spike-In control Mix (Thermo Fisher Scientific, #4456740). About 400 ng of total RNA was ribosomal depleted and fragmented and reverse transcribed. Samples underwent end repair and dA-Tailing step followed by ligation of NEBNext adapters. The libraries were pooled and sequenced on the Illumina NovaSeq 6000 with paired-end 150 bp at the University of Michigan core facility or AZENTA.

RNA was extracted from 4 mL of fractionated plasma using a plasma/serum circulating and exosomal RNA purification kit (Norgen Biotek, 42800) according to the manufacturer’s protocol with the following modifications. After fractionated plasma samples were lysed at 60 °C for 10 min and mixed with ethanol in step 2 of the procedure, 10 μL of 10⁶ times diluted ERCC RNA spike-in control mix (Thermofisher, 4456740) was added into each denatured plasma fraction sample (i.e. after combining the plasma fraction samples with denaturing solution in step 1) on ice as an external RNA control for normalization. These ERCC RNA-spiked in plasma fraction samples were followed by centrifugation at 1000 RPM for 2 min. After that point, we followed the manufacturer’s protocol and eluted RNA in 100 μL. To digest trace amounts of contaminating DNA, we treated the RNA with 10X Baseline-ZERO DNase (Lucigen, DB0715K). DNase I treated RNA samples were purified and further concentrated using RNA clean and concentrator-5 (Zymo Research, R1014) according to the manufacture’s protocols. Final eluted RNA was aliquoted and stored at −80 °C immediately.

Total RNA was isolated using RNeasy Mini Plus kit (Qiagen, USA) according to the manufacturer’s instructions. RNA concentration was measured on a NanoDrop (Thermo Fisher, USA). RNA samples from LNCaP-19 cells and PC-3 cells co-cultured with mature osteoclasts or control cells (RAW 264.7 cells) were quantified and checked for contamination using (Dropsense96, Trinean). After normalization for equal input (500 ng in 5 ul), samples were spiked with ERCC RNA Spike-In Control Mix (ThermoFisher). Libraries were prepared using QuantSeq 3′ mRNA-Seq (FWD) Library Prep Kit for Illumina (Lexogen) including individual indexing for multiplex sequencing. Finalized libraries were quality checked with capillary electrophoresis (Fragment Analyzer) and quantified with Picogreen (ThermoFisher). The libraries were normalized and pooled before NextSeq500 sequencing (Illumina).