All animal procedures described in this study were approved by the Institutional Animal Care and Use Committee (IACUC) (#6826170118) and performed in full compliance with the recommendations of Federal Law 11.794 (2008), the Guide for the Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation (CONCEA) and the ARRIVE guidelines.
Ten- to 12-week-old male C57BL/6 mice were anesthetized with an intraperitoneal injection of 100 µl of anesthetics (2.5% ketamine/0.26% xylazine in saline) prior to surgery-induced hind limb ischemia. Then, the left femoral artery was exposed and cauterized at its origin between the external iliac artery branch and its bifurcation into the saphenous and popliteal arteries  on day 0. The blood flow of the limbs was examined using a laser Doppler imaging (LDI) system (moorLDI2-IR; Moor Instruments, Axminster, United Kingdom). Ischemic mice were orally administered tramadol (20 µl of 24 mg/ml) in the morning and evening to manage pain during the first 2 days after surgery. Ischemic gastrocnemius muscles were collected on postsurgery days 2, 4, and 7 to analyze the pericyte population.
The gastrocnemius muscles were collected and immediately digested with 2 ml of 0.2% collagenase (#C0130, Sigma-Aldrich Co., St. Louis, MO, USA) in Dulbecco’s Modified Eagle’s Medium (DMEM) at 37°C for 40 min. Tryptic action was then halted by adding the same volume (2 ml) of stop solution [50% fetal bovine serum (FBS)/50% DMEM]. Samples were then sequentially passed through 70-µm and 40-µm cell strainers to remove tissue debris. Cells were washed with PBS once. Samples were then incubated with Fc blockers (#14-0161-81, anti-CD16/CD32 antibody; Thermo-Fisher Scientific, Waltham, MA, USA) at a dilution of 1:1000 in 1% FBS/phosphate-buffered saline (PBS) on ice for 10 min and washed once with PBS. Cells were stained with the following antibodies diluted in PBS: anti-CD45-Pacific blue (clone 30-F11, #MCD4528, Thermo-Fisher Scientific, Waltham, MA, USA; 1:100), anti-CD31-PE/Cy7 (clone MEC13.3, #102524, Biolegend, San Diego, CA, USA; 1:100) and anti-CD146-PerCP/Cy5.5 (clone ME-9F1, #102524, Biolegend; 1:50) on ice for 30 min. After washes with PBS, the cells were stained with the fixable viability dye 780 (#565388, BD Pharmingen, San Jose, California, USA) at a dilution of 1:1,000 in PBS on ice for 30 min. After another wash, cells were incubated with Fixation/Permeabilization solution (Cytofix/Cytoperm, #554722, BD Pharmingen) for 20 min at room temperature and washed with Perm/Wash buffer (554723, BD Pharmingen). An anti-Nestin-PE antibody (clone 307501, #MA5-23574, Thermo-Fisher Scientific, Waltham, MA, USA; 1:10) diluted in Perm/Wash buffer was used to stain the cells on ice for 30 min. After washes with Perm/Wash buffer, the cells were fixed with 1% paraformaldehyde in PBS for 15 min. Then, after a PBS wash, the cells were resuspended in 2% FBS in PBS and incubated at 4°C overnight. The cell suspensions from each gastrocnemius muscle were analyzed with a BD LSRFortessa flow cytometer (BD, San Jose, CA, USA) after voltage adjustment using unstained and single-color controls to ensure proper compensation and analysis. In addition, fluorescence minus one (FMO) tubes were used to allow proper gate setting. The number of events acquired per sample ranged from 0.3 to 1.2 million. All flow cytometry data were analyzed using FlowJo Cytometry software (v.10; BD Biosciences). For the flow cytometry analysis, an FSC-A vs. SSC-A dot plot was used to exclude cell debris, and an FSC-A vs. FSC-H dot plot was used to exclude cell clumps and doublets. The number of gated singlet cells obtained after this procedure ranged from 163,000 to 772,000 per sample. Live cells were selected as the viable dye-negative population. Only the CD45- population was subjected to further analyses to exclude nonlymphocytic cells. The pericytes were then identified as a CD31-CD146+ cell population. Next, the type I (Nestin-) and type II (Nestin+) pericyte subpopulations were gated. Discrimination of positive events using this gating strategy was based on the FMO samples.
FACS (fluorescence-activated cell sorting)
Each muscle was digested individually as described above in the flow cytometry section. Multiple digested muscles were combined and filtered through cell strainers. The cells were washed once with PBS and once with wash buffer [3% FBS/1:5000 RNaseOUT (#10777019, Thermo-Fisher Scientific) in PBS]. Samples were incubated with Fc blockers (1:1,000 diluted in wash buffer) on ice for 10 min and then washed with wash buffer. The cells were stained with the following antibodies (diluted in wash buffer) on ice for 30 min: anti-CD45-Pacific blue (clone 30-F11, #MCD4528, Thermo-Fisher Scientific; 1:100), anti-CD31-PE/Cy7 (clone MEC13.3, #102524, Biolegend; 1:100) and anti-CD146-PerCP/Cy5.5 (clone ME-9F1, #102524, Biolegend; 1:50). The fixable viability dye 780 was used to stain cells at a 1:1,000 dilution in PBS on ice for 30 min. The cells were washed with wash buffer, and the samples were resuspended in wash buffer for cell separation using a BD FASCAria II cell sorter. Compensation adjustment was performed using compensation beads (#552843 and #552845, BD Pharmingen), and FMO controls were used to enable proper gate setting. The sorted cells were collected in collection buffer (10% FBS/1:500 RNaseOUT in PBS) and analyzed to conﬁrm the purity.
Pooled muscle cell suspensions from different mice were used for staining and sorting to ensure that the number of sorted cells was sufficient to provide a sufficient amount of RNA required for the transcriptome analyses. In the nonischemia group, 10 gastrocnemius muscles collected from 5 mice were digested and pooled as one cell suspension sample for subsequent staining and sorting because of the low event number of total cells and pericytes. In ischemic muscles, the event numbers of total cells and pericytes were increased; therefore, two digested muscles were pooled into one cell suspension for further staining and cell sorting.
Total RNA extraction
Cells sorted by FACS were centrifuged at 3,000 xg for 10 min at 4°C, and the cell pellet was lysed with 300 µl of TRIzol (#15596026, Thermo-Fisher Scientific, Waltham, MA, USA) with pipetting. After mixing with chloroform and centrifugation, the RNA-containing aqueous supernatant was transferred to a new tube. Next, 0.5 µl of 20 µg/µl glycogen (#10814010, Thermo-Fisher Scientific, Waltham, MA, USA), 40 µl of 2 M sodium acetate and 500 µl of 100% ethanol were sequentially added to facilitate RNA precipitation. This crude RNA extract was stored at -80°C. For further purification, this sample underwent a series of clean-up steps, DNaseI treatment, and RNA elution according to the instructions provided with the Ambion RecoverAll Nucleic acid Isolation Kit (#AM1975, Thermo-Fisher Scientific, Waltham, MA, USA). All the RNA samples were stored in LoBind® microcentrifuge tubes (#22431021, Eppendorf AG, Hamburg, Germany).
Reverse transcription and cDNA amplification
RNA reverse transcription was performed based on the method described by Picelli et al. . Briefly, for each reaction, 2.3 µl of RNA were mixed with Oligo-dT30VN primers and dNTPs, heated at 72°C for 3 min, and then chilled immediately on ice. TSO primers mixed with reverse transcriptase were added to perform cDNA synthesis. Next, the cDNA templates were amplified by ISPCR primers and high-fidelity DNA polymerase. The following reagents were used: SuperScript™ IV Reverse Transcriptase (#18090050, Thermo-Fisher Scientific, Waltham, MA, USA), RNaseOUT (#10777019, Thermo-Fisher Scientific, Waltham, MA, USA), KAPA HiFi HotStart ReadyMixPCR Kit (#KK2602, Kapa Biosystems, USA), 5 M Betaine (#B0300-1VL, Sigma-Aldrich Co., St. Louis, MO, USA), and 1 M MgCl2 (#M1028-10X1ML, Sigma-Aldrich Co., St. Louis, MO, USA). AMPure XP magnetic beads (#A63880, Beckman Coulter, Brea, CA, USA) were used to purify the cDNAs. The concentrations of cDNAs were then determined using Qubit DNA HS assay (#Q32854, Thermo-Fisher Scientific, Waltham, MA, USA).
RNA-seq library preparation and data analysis
A Nextera XT DNA Library Prep Kit was used to construct cDNA libraries. Sequencing was performed using a NextSeq 500/550 High-Output v2.5 Kit (150 cycles) (#20024907, Illumina, San Diego, CA, USA) at CEFAP GENIAL (Genome Investigation and Analysis Laboratory; http://cefap.icb.usp.br/core-facilities/genial-genome-investigation-and-analysis-laboratory/). Raw data were first examined using FastQC (Version 0.11.8). After trimming with Trimmomatic (Version 0.39)  using the default settings, the data were analyzed with FastQC again. Rsubread (Version 1.28.1/R Version 3.4) was used for both alignment and generation of counts . The count per gene database was then inputted to DESeq2 (Version 1.24.0) for differential expression analyses . The time-course pattern analysis was performed using the Short Time-series Expression Miner (STEM) program . Statistically significant differences reported by STEM are based on a correlation test at a significance level of 0.05 followed by Bonferroni correction of the p value. The GO enrichment analysis was conducted using the software package "ClusterProfiler" version 3.12.0 . Heatmaps were generated using the software package “pheatmap” using rlog transformed counts calculated by “DEseq2”.