Idt for rna ud indexes set a

Mouse back skin from CR, GF, CR×CR F₁, and GF×GF F₁ was collected and fixed overnight in 4% paraformaldehyde (Fisher AAJ19943K2) at 4°C followed by paraffin embedding. Laser capture microdissection (LCM) was performed using the LMD 7000 system (Leica Microsystems). FFPE mouse skin was processed and cut onto a polyethylene naphthalate (PEN) slide designed for LCM processing (Leica 11505158). At least 1,000 SGs or 1,000,000 μm² of tissue was isolated to obtain enough material for RNA extraction. SG RNA was extracted from post-LCM tissue using a Qiagen All Prep DNA/RNA FFPE Kit (Qiagen 80234). RNA concentration was measured by Qubit fluorometric quantification (ThermoFisher Qubit 2.0 Fluorometer) and RNA quality measured via BioAnalyzer (Agilent 2100 Bioanalyzer Instrument). cDNA libraries were prepared using Illumina Stranded Total RNA Prep with Ribo-Zero Plus Kit (Illumina 20040529) with IDT for Illumina RNA UD Indexes, Set A (Illumina 20040553). Libraries were assessed for cDNA quantity and library quality using Qubit and BioAnalyzer. As necessary, an extra bead wash step was performed to remove excess primer dimers in the library and purify samples further. Samples were then pooled and sequenced on a Nextseq 550 using a NextSeq 500/550 High Output Kit v2.5 (150 Cycles) (Illumina 20024907).

To prepare RNA-seq libraries, we used Illumina Stranded mRNA Prep Kit (#20040532, Illumina, San Diego, CA, USA) and IDT® for Illumina® RNA UD Indexes Set A (#20040553, Illumina, San Diego, CA, USA) according to manufacturer's instructions. The quantity and quality of the RNA-seq libraries were measured using Qubit 4 Fluorometer, NanoDrop One spectrophotometer, and 2100 Bioanalyzer Instrument. The RNA-seq libraries were multiplexed and then sequenced from both ends on the Illumina Novaseq 6000 with a 2 × 150 paired end (PE) configuration. The next-generation sequencing (NGS) was carried out in the Advanced Genomics Core at the University of Michigan. The FASTQ files obtained in this study were uploaded to the BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) and are available under the accession number PRJNA1015283.

hCOs were treated with nifedipine or DMSO (vehicle control) for 8 hours (n = 5 hCOs per condition). hCOs were washed with PBS, lysed, and stored at −80 degrees. RNA extraction was conducted using the NucleoMag 96 RNA kit (Macherey-Nagel) on the EpMotion 5075t. RNA was quantified using the Qubit RNA High Sensitivity Assay (Invitrogen). RNA integrity was assessed using the 4200 TapeStation with the High Sensitivity RNA ScreenTape kit (Agilent). Samples were normalized to ≤100 ng RNA using nuclease free water in a total of 25μl. Poly(A)-enriched RNA libraries were prepared using the Illumina Stranded mRNA Prep, Ligation (96 Samples), and the IDT for Illumina RNA UD Indexes Set A (Integrated DNA Technologies). Libraries were quantified using the Quant-iT dsDNA broad range (BR) assay kit (Invitrogen). The quality of libraries was assessed using the 4200 TapeStation with the D1000 ScreenTape kit (Agilent). Single-end 100bp sequencing was conducted on the NovaSeq 6000 (Illumina).