C1000 touch thermos cycler cfx96 real time system

DNA from iPSC‐ and iNPC‐derived young and old astrocytes was extracted with the DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer's protocol. Telomere length of iPSC and iNPC‐derived astrocytes was measured using the Absolute Human Telomere Length quantification qPCR assay Kit (ScienCell Research Laboratories, San Diego, CA, USA) according to the manufacturer's instructions. A single copy reference (SCR) primer set recognises and amplifies a 100‐bp‐long region on human chromosome 17 and is used as a reference for data normalisation. The reference genomic DNA sample with known telomere length was used as reference for calculating the Telomere Length of iPSC (Table S5) and young and old iNPC‐derived astrocytes. qPCR products were performed on a C1000 Touch™ thermos Cycler CFX96™ Real‐Time System (Bio‐Rad), and qPCR data were analysed using CFX Manager™ software (Version 3.1) (Bio‐Rad) and GraphPad Prism (Version 8).

Following quantification, 1 μg RNA (iNeurons samples) or 2 μg RNA (Drosophila samples) was converted to cDNA using BioScript Reverse Transcriptase (Bioline). qRT–PCR primers were designed using Origene or Primer-BLAST and validated using a 1 in 4 serial template dilution series (standard curve with R² > 0.97). qRT–PCR reactions were performed in duplicate using the Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies) on a C1000 Touch™ thermos Cycler CFX96™ Real-Time System (BioRAD) using an initial denaturation step, 45 cycles of amplification (95 °C for 30 s; 60 °C for 30 s; 72 °C for 1 min) prior to recording melting curves. qRT–PCR data was analysed using CFX Manager™ software (Version 3.1) (BioRAD) and GraphPad Prism (Version 7). The sequences of qPCR primers are provided in Additional file 1.