Smarter ultra low rna kit for sequencing

Bone marrow cells were harvested from H2afy^+/+ and H2afy^−/− mice of 5–12 weeks of age. Cells were enriched for c-Kit⁺ cells using CD117 MicroBeads on the autoMACS Pro Separator (Miltenyi Biotec). Subsequently, cells were stained according to the staining scheme described above, and BLPs were sorted by FACS (Moflo, Beckman Coulter) directly into the lysis buffer of NucleoSpin RNA XS kit (MACHEREY-NAGEL). RNA was isolated per manufacturer’s instructions. Per sample, RNA equivalent of 5,000–15,000 BLPs from 2–3 mice were pooled. Total RNA integrity and concentration were assessed using a 2100 Bioanalyzer and an RNA 6000 Pico kit (Agilent). Library preparation was performed with 10–50ng of total RNA with a Bioanalyzer RIN score greater than 8.0. Double stranded-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer’s protocol. cDNA was fragmented using a Covaris E220 sonicator using duty cycle 10, intensity 5, cycles/burst 200, time 180 s. cDNA was blunt ended, had an A base added to the 3′ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases.

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.