Fragment analyzer

The total RNA was checked for quality by Fragment Analyzer (Advanced Analytical). High-quality samples (RQN > 8) were used to prepare the library using the TruSeq RNA Library Prep Kit v2 (Illumina). Libraries were checked for quality and quantified using the Fragment Analyzer and Qubit (ThermoFisher Scientific), respectively, before being sequenced on Nextseq 500 (Gene expression core facility, EPFL). Two biological replicates were performed for each condition.

VDJ, 5′-mRNA, and feature barcode libraries were prepared using the 10× Chromium System (10X Genomics, Pleasanton, CA) according to the manufacturer’s instructions. The C Chromium Next GEM Single Cell 5′ Kit v2 (10X Genomics, PN-1000263), Chromium Single Cell Human BCR Amplification Kit (10X Genomics, PN-1000253), and 5′ Feature Barcode Kit (10X Genomics, PN-1000256), Library Construction Kit (10X Genomics, PN-1000190), and Dual Index Kit TT Set A (10X Genomics, PN-1000215), Dual Index Kit TN Set A (10X Genomics, PN-1000250) were used. All libraries were quantified by using Qubit 3.0 (Thermo Fisher), Fragment Analyzer, and qPCR. Sequencing was performed on a Hiseq 2500 platform running Rapid SBS Kit V2 2x100bp kit (Illumina, FC-402–4021), with 10 cycles for the i7 index and i5 index. Average sequencing depth aimed for the mRNA library is 20,000 read pairs per cell, 5000 read pairs per cell for the VDJ libraries, and 5000 read pairs for feature barcode libraries.

RNA was isolated from PBMC samples with the RNeasy kit including on-column DNase I digestion, as directed (Qiagen, Valencia CA). RNA quantity was determined with Qubit high sensitivity RNA assay (Thermo Fisher Scientific, Waltham, MA) and RNA integrity was determined with an AATI Fragment Analyzer at the Cornell Genomics Facility (Cornell University, Ithaca NY), with acceptable scores ≥ 7.7. These RNA samples were used to generate transcriptome (RNA-Seq) libraries with TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA). Libraries were assessed for insert size (260 bp) and DNA concentration (Qubit high sensitivity DNA assay, Thermo Fisher Scientific). After normalization, libraries were multiplexed and sequenced on an Illumina NextSeq500 at the Cornell Genomics Facility. The number of samples multiplexed per run was 12. The RNA-Seq dataset is available in the NCBI Gene Expression Omnibus repository as series GSE101117.

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).

Construction of the RNAseq libraries and sequencing on the Illumina NovaSeq 6000 were performed at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. After DNase digestion, purified total RNAs were analyzed on a Fragment Analyzer (Agilent) to evaluate RNA integrity. The total RNAs were converted into individually barcoded polyadenylated mRNAseq libraries with the Kapa HyperPrep mRNA kit (Roche). Libraries were barcoded with Unique Dual Indexes (UDI’s) which have been developed to prevent index switching. The adaptor-ligated double-stranded cDNAs were amplified by PCR for 8 cycles with the Kapa HiFi polymerase (Roche). The final libraries were quantitated with Qubit (Thermo Fisher) and the average cDNA fragment sizes were determined on a Fragment Analyzer. The libraries were diluted to 10 nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system (Bio-Rad) for accurate pooling of barcoded libraries and maximization of number of clusters in the flowcell.

Following dissociation, cells were diluted to 110 cells/µl in PBS/0.01% BSA. Drop-seq was performed as described in Macosko et al.^{79 (link)}. Briefly cells and barcoded beads (Chemgenes, 132 beads/µl in lysis buffer) were run on an aquapel-treated microfluidic drop-seq device (FlowJEM) for co-encapsulation in nanoliter-sized droplets. After droplet-breakage, reverse transcription and Exonuclease I treatment, beads were counted, and 2500 beads were apportioned per PCR tube for cDNA amplification. The amplified cDNA libraries were purified using SPRI beads (Beckman Coulter) and quantified on a Fragment Analyzer (Agilent). Tagmentations were performed using the Illumina Nextera XT kit (Illumina), and the resulting libraries were purified in two consecutive rounds of SPRI beads-based size selection (0.6x beads to sample ratio followed by 1x beads to sample ratio). The size and concentration of the final libraries were measured on a Fragment Analyzer and a Qubit Fluorometer (Thermo Fisher), respectively. Libraries were sequenced on an Illumina Nextseq500 instrument.

Total RNAs were run on a Fragment Analyzer (Agilent, Santa Clara, CA) to evaluate RNA integrity. The RNAseq libraries were constructed with the TruSeq Stranded mRNA Sample Prep kit (Illumina, San Diego, CA). Briefly, polyadenylated messenger RNAs (mRNAs) were enriched from 500 ng of high-quality deoxyribonucleic acid (DNA)-free total RNA with oligodT beads. The mRNAs were chemically fragmented, annealed with a random hexamer, and converted to double stranded cDNAs, which were subsequently blunt-ended, 3'-end A-tailed, and ligated to indexed adaptors.
Each library was ligated to a uniquely dual indexed adaptor (unique dual indexes) to prevent index switching. The adaptor-ligated double-stranded cDNAs were amplified by polymerase chain reaction (PCR) for eight cycles with the Kapa HiFi polymerase (Roche, CA) to reduce the likeliness of multiple identical reads because of preferential amplification. The final libraries were quantified with Qubit (ThermoFisher), and the average library fragment length was determined on a Fragment Analyzer. The libraries were diluted to 10 nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system (Biorad, Hercules, CA) for accurate pooling of the barcoded libraries and maximization of number of clusters in the flow cell.