Dragen bio it

We plated HUVECs in the bottom compartments of 24-well transwell plates (0.4 μm pore size, Corning) along with the following in the top compartments: 1) cell-free (n = 6 wells); 2) naive BMDMs (n = 6 wells); or 3) CYR61-treated BMDMs (0.5 μg/mL, 24 h, n = 6 wells). After co-incubation for 24 h, we washed HUVECs twice with PBS, then used TRIzol (Ambion) and RNeasy Plus Mini Kit (Qiagen) to extract total RNA following the manufacturer’s recommended protocol. We quantified the quality and quantity of the RNA samples with an Agilent Bioanalyzer or 4200 Tapestation. All 18 samples had RIN ≥ 8.9. We prepared ds-cDNA using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per the manufacturer’s protocol. Then, we fragmented cDNA, blunt ended the cDNA, added an A base to the 3′ ends, and then ligated Illumina sequencing adapters to the ends. We amplified the ligated fragments and incorporated unique dual index tags. We then sequenced the fragments on an Illumina NovaSeq 6000 using paired end reads extending 150 bases at the McDonnell Genome Institute at Washington University School of Medicine in St. Louis. Reads were then aligned and quantitated to the Ensembl release 101 primary assembly with an Illumina DRAGEN Bio-IT on-premise server running version 3.9.3-8 software.

For RNA sequencing, we first removed ribosomal RNA by an RNase-H method using RiboErase kits (Kapa Biosystems). Then, we fragmented mRNA and then reverse transcribed mRNA to cDNA using SuperScript III RT enzyme (Life Technologies) and random hexamers following the manufacturer’s instructions. We performed a second strand reaction to yield ds-cDNA. Then, we blunt-ended the cDNA, added an A base to the 3′ ends, and then ligated Illumina sequencing adapters to the ends. We amplified ligated fragments and incorporated unique dual index tags. We sequenced fragments on an Illumina NovaSeq 6000 using paired end reads extending 150 bases at the McDonnell Genome Institute at Washington University School of Medicine in St. Louis. RNA sequencing reads were then aligned and quantitated to the Ensembl release 101 primary assembly with an Illumina DRAGEN Bio-IT on-premise server running version 3.9.3-8 software. We identified 1 of the 8 control samples to be an outlier and excluded it from further analyses.

We incubated HUVECs with recombinant human CYR61 (PeproTech Cat# 120-25) at 4 concentrations: 0.00, 0.05, 0.50, and 5.00 μg/mL for 24 h (n = 5 wells/group). After incubation, we washed cells twice with PBS, then used TRIzol (Ambion) and RNeasy Plus Mini Kit (Qiagen) to extract total RNA following the manufacturer’s recommended protocol. We quantified the quality and quantity of the RNA samples with an Agilent Bioanalyzer or 4200 Tapestation. All 20 samples had RIN ≥ 9.7. We prepared ds-cDNA using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) following the manufacturer’s protocol. Then, we fragmented cDNA, blunt ended the cDNA, added an A base to the 3′ ends, and then ligated Illumina sequencing adapters to the ends. We amplified the ligated fragments and incorporated unique dual index tags. We then sequenced the fragments on an Illumina NovaSeq 6000 using paired end reads extending 150 bases at the McDonnell Genome Institute at Washington University School of Medicine in St. Louis. Sequenced reads were then aligned and quantitated to the Ensembl release 101 primary assembly with an Illumina DRAGEN Bio-IT on-premise server running version 3.9.3-8 software.