High capacity kit

Total RNA of the samples collected after 30- and 60-days post-surgery were macerated in liquid nitrogen and isolated using TRIzol^TM reagent (Invitrogen, Waltham, MA, USA) for RNA isolation, following the manufacturer’s instructions. The RNA was converted into cDNA from 1.5 µg of total RNA using the high-capacity kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The TaqMan assays used are described in Table 3, were purchased from Applied Biosystems. The reactions were performed in triplicate with TaqMan Gene Expression Master Mix (Applied Biosystems, Waltham, MA, USA). The entire qPCR procedure was performed according to the protocol previously described [20 (link),21 (link)]. The Gapdh was used as the control for data normalization and validated using the BestKeeper software. The PCL group was used as a calibrator and the results were calculated using the 2^−∆∆Ct method.

CHLA-01 and CHLA-01R RNA was extracted using TRIzol^TM reagent (Thermo Fisher Scientific, Grand Island, NY, USA; Cat# 15596018) and cDNA was synthesized using a High-Capacity Kit (Thermo Fisher Scientific, Grand Island, NY, USA; Cat# 4368814) according to the manufacturer’s protocol. Relative levels of mRNA were determined by real-time quantitative PCR using TaqMan Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA) or PowerUp SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA). Reactions were performed on ViiA™ 7 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Data were analyzed using the 2^−ΔΔCT method with GAPDH as an endogenous control. The probes and primers used in qRT-PCR are listed in Table S1.
Samples of CHLA-01R cells were transfected in triplicate with control siRNA or Msi1 siRNA, as described above. RNA samples were obtained as described above and analyzed by RNA-seq. Samples were processed for RNA-seq according to the manufacturer’s instructions (Illumina, San Diego, CA, USA) and sequenced on a HiSeq-3000 machine by the UTHSCSA Genomic Facility.

Isolation of total RNA was done using GenElute™ Mammalian Total RNA Miniprep Kit (www.sigmaaldrich.com, accessed on 5 April 2021), following the DNase I (RNase-free) treatment (www.neb.com, accessed on 5 April 2021). cDNA was synthesized using the High Capacity Kit (www.thermofisher.com, accessed on 5 April 2021). RNA quality and DNA integrity were checked using control primers for Gapdh.

For quantitative real-time TaqMan PCR, 250 ng total RNA was reversely transcribed into cDNA using a high capacity kit (Thermo Fisher Scientific, Schwerte, Germany) and subsequently diluted to a final contraction of 1.25 ng/µL. Subsequently, 5 µL gene expression Mastermix (Thermo Fisher Scientific, Schwerte, Germany) and 0.5 µL of TaqMan gene expression assay purchased from Thermo Fisher Scientific (Schwerte, Germany) were used in a final volume of 10 µL including 1 µL of cDNA template (performed in duplicates). As an internal control, for each cDNA, the gene expression of the housekeeping gene CDKN1B was quantified and verified that it was not affected by treatments. Gene expression of target genes was normalized to the determining gene expression level of CDKN1B using the formula 2^−ΔCt. Expression data were displayed as mean values for untreated and treated samples and visualized as spaghetti plots for each patient separately using GraphPad Prism 6.05 (GraphPad Software, La Jolla, CA, USA). Statistical comparison of two groups was performed using the ratio paired t-test with p-values < 0.05 considered as statistically significant.

Four NURR1-KO and seven WT littermates mice were euthanized by inhalation of isoflurane and brains were removed. The brain areas (i.e. SN and VTA) were manually dissected from 300 µm-thick coronal sections cutter by vibratome (Bregma −2.92 mm, Interaural 0.88; Bregma −3.88 mm, Interaural −0.08). All samples were rapidly frozen in 2-methylbutane in dry ice and total RNA was isolated by extraction with the Pure Link RNA Mini Kit (Thermo Fisher Scientific, US) according to the manufacturer’s instructions. Total RNA was reverse-transcribed to complementary DNA (cDNA) at a final concentration of 20 ng/μl using the High Capacity Kit (Thermos Fisher Scientific, Waltham, MA, USA). Gene expression analysis was performed by real-time PCR using Applied Biosystems’ TaqMan gene expression products (Thermos Fisher Scientific, Waltham, MA, USA). Transcriptional expression was normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene. For primers and probes, Applied Biosystems’ TaqMan® Assay-on-demand-TM gene expression products were used (GAPDH; Mm99999915_g1, TH; Mm00447557_m1). Expression levels of target genes were calculated by the normalized comparative cycle threshold (Ct) method (2^-ΔCt).

DRGs were collected from naive or SNI Cx3cr1^GFP/+ mice (DRGs from eight mice were pooled). As described above, the tissues were digested and filtered through a cell strainer. Samples were then centrifuged, and the supernatant was discarded. Cellular pellet was resuspended in a solution containing cell surface markers (CD11b, CD45, and Live/Dead), stained for 10 min at room temperature, and then further sorted as macrophages (CX3CR1⁺CD11b⁺) in a FACS Aria III sorter. The full-gating strategy used to perform cell sorting and all flow cytometry experiments from sensory ganglia is depicted in Figure 8—figure supplement 1. Sorted cells were submitted to RNA extraction (using the RNeasy Micro Kit - Qiagen), reverse-transcribed with High Capacity Kit (Thermo Fischer Scientific), and analyzed by RT-PCR with a Step One Real-time PCR system as described above (Applied Biosystems).