Nanodrop 2000c system

The messenger RNA (mRNA) expression levels of Drp1, Fis1, Opa1, Mfn1, Mfn2, and Actb (encoding β‐actin) were measured using qRT‐PCR. Total mRNA was isolated from the hippocampus using Trizol (Invitrogen) and quantified with a NanoDrop 2000c system (Thermo Fisher Scientific). The sequence details of individual pairs of primers are shown in Table 1; the experimental protocol has been described previously.²⁸ The PCR consisted of 40 cycles of 5 s at 95°C and 15 s at 60°C. The relative mRNA levels were measured using the 2^{−Δ(Δ CT)} method and normalized to Actb levels.

Quantitative expression of miR-8b and related genes was monitored using qPCR. Total RNA was extracted from planarians with Trizol according to the manufacturer’s instructions at different time points (0, 1, 3, 5, 7, and 10 days). Washed RNA was dissolved in 20 μl nuclease-free water and the concentration was determined by the NANODROP 2000C system (Thermo Fisher Scientific). A 15–20 bp poly(A) tail was added to all RNA using Poly(A) polymerase (NovoBiotec, PAP5 104H) followed by first strand synthesis (Roche, 04379012001) using an oligo(dT) linked random primer adapter. The control treated fragments and perturbing treated fragments were detected at different time after amputation by qPCR. miR-8b F-primer sequence is: 5′-TAATACTGTCAGGTAAGAAT-3′, R-primer is 5′-GCGAGCACAGAATTAATACGACTC-3′, β-actin F-primer sequence is: 5′-ACACCGTACCAATCTATG-3′ and R-primer: 5′-GTGAAACTGTAACCTCG-3′, Djndk F-primer sequence is: 5′-TCACAAACTCCACCGCAGTACTTT-3′ and R-primer: 5′-GGTATGGATTAGCATTATTGAATTGTG-3′. cDNA was synthesized using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qRT-PCR was performed using SYBR Green PCR Master Mix (Roche) and internal controls were used as the β-actin gene. Relative RNA abundance was repeated three times and analyzed using the Roche LightCycler 48Ⅱwith the comparative Ct method (2−△△ct).

Cells were resuspended in Trizol and the protein fraction was further washed with 96% ethanol. The proteins were precipitated with 2-propanol and the pellet was washed three times with 0.3 M Guanidine-HCl in 96% ethanol. The pellet was resuspended in 1:1 solution of 1% SDS:8 M Urea in 1 M Tris-HCl. All the reagents for protein extraction were purchased from Sigma Aldrich, St. Louis, USA. After five cycles of 15 s of sonication steps, the samples were stored at −80°C. Equal amounts of proteins, quantified using the Nanodrop 2000c system (Thermo Fisher Scientific, Waltham, MS, USA), were separated in precast Mini-Protean TGX gels (Bio-Rad, Hercules, CA, USA) and transferred onto 0.45 μm nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% nonfat dry milk in Tris-buffer saline with Tween 20 (Invitrogen, Carlsbad, CA, USA) (TBST), the membranes were incubated for 2 h at room temperature with a primary antibody for Caspase-3 (Abcam, Cambridge, UK), Cytochrome C (Abcam, Cambridge, UK), and GAPDH (Abcam, Cambridge, UK), with stripping steps between primary antibody incubations. After washing with TBST, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase. The signals were detected after adding the chemiluminescence substrate (Invitrogen, Carlsbad, CA, USA) [8 (link),20 (link)].

Total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer’s instructions. Washed RNA was dissolved in 20 μl nuclease-free water and the concentration was determined using the NANODROP 2000C system (Thermo scientific).
Illumina’s TruSeq RNA Preparation Kit was used to purify 15-40 nt small RNAs from total RNA. Size selection and quality control were performed by BGI, Hong Kong. Libraries for each time point were sequenced on an Illumina HiSeq4000 sequencer (single-end, 50 bp). The raw reads were filtered according to the following criteria: 1) more than four bases had a quality score below 10 or more than six bases had a quality score below 13. 2) Reads containing homo-polymers, such as poly-A. 3) Reads with 5′ adapter contaminants or without 3′ adapter sequence. 4) Reads without insert tag. 5) Reads with a length below 18 nt. Sequence data can be accessed at NCBI’s Sequence Read Archive under the accession PRJNA549868.