Coralload concentrate

The methylation status of LINE-1 was measured by pyrosequencing. The primer sequences and PCR conditions have been previously described in detail [30 (link), 31 (link)]. Briefly, PCR was carried out in a 25 μL reaction mix containing 50ng bisulfite-converted DNA, 1x Pyromark PCR Master Mix (Qiagen, Valencia, CA), 1x Coral Load Concentrate (Qiagen) and 0.2 uM forward and reverse primers, using the following PCR program: 95°C for 15 minutes, then 44 cycles of 95°C for 30 seconds followed by 56°C for 30 seconds and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes. The biotinylated PCR products were purified and converted into single-strands to act as a template in the pyrosequencing reaction as recommended by the manufacturer of the Pyrosequencing Vacuum Prep Tool (Qiagen). Then, 0.3 nmol/L of pyrosequencing primer was annealed to the purified single-stranded PCR product and pyrosequencing was conducted on a PyroMark Q96 MD (Qiagen). We used non-CpG cytosine residues as internal controls to verify efficient sodium bisulfite DNA conversion and universal unmethylated and methylated DNAs (Zymo Research) were used as experimental controls. The intra- and inter-assay coefficients of variation were 0.7% and 1.4%, respectively.

DNA was bisulfite-converted using the EpiTect Plus DNA bisulfite kit (Qiagen) and stored at −20 °C until utilized. PCR amplification of IL6 promoter was performed using 2x PyroMark PCR Master Mix and 10x CoralLoad Concentrate (Qiagen), 0.2 µM of each primer, 1 µL of converted-DNA and nuclease-free water to a final volume of 25 µL. Primers used for DNA methylation analysis and PCR cycling conditions are shown in Table S1 of the Supplementary Information File. Pyrosequencing reaction was run on a PyroMark Q96ID (Qiagen) and CpGs methylation analysis was conducted by the PyroMark CpG software (Qiagen). A triplicate was generated for each PCR. Methylation for each amplicon was calculated as the median of methylation status of each analyzed CpG. Differences in methylation pattern across samples and controls were calculated by two-tailed student T-test, considering p-values < 0.05 as significant.

A custom pyrosequencing assay was designed to target the top differentially methylated position from the BeadChip data. The assay was designed using PyroMark Assay Design software 2.0 (Qiagen). Ten nanograms of template bisulfite‐converted DNA was added to 1× PyroMark PCR Master Mix (Qiagen), 1× CoralLoad Concentrate (Qiagen), and 0.2 μM PCR primer set (Qiagen). The solution was made up to 25 μl with RNase‐free water (Qiagen) and was amplified in a thermocycler (DNA Engine Dyad; MJ Research) using polymerase chain reaction (PCR) conditions recommended by the manufacturer (Qiagen).
The remaining PCR product (10–20 μl) was agitated at 1,400 revolutions per minute for 10 minutes with 2 μl streptavidin–Sepharose High Performance Beads (GE Healthcare) and 40 μl PyroMark Binding Buffer (Qiagen) made up to 80 μl with Milli‐Q water. The samples were applied to the vacuum handset of the PyroMark Q24 workstation and washed with 70% ethanol, PyroMark denaturation solution, and PyroMark wash buffer (Qiagen) before being added to 0.3 μM sequencing primer diluted to 25 μl in annealing buffer (Qiagen). To allow the biotin‐labeled DNA strand to anneal to the primers, the solution was heated to 80°C for 2 minutes, then allowed to cool to room temperature for 5 minutes. Pyrosequencing reactions were performed using a PyroMark Q24 and PyroMark Gold Q24 Reagents (Qiagen).

RNA was isolated from ASCs and neuronal cells (appropriate passages) using a RNeasy Mini Kit (Qiagen, cat. no. 74104). The purity of the RNA was confirmed using the 260/280 nm absorbance ratio, and the RNA was quantified using a Bio Photometer D30 (Eppendorf). The isolated RNA was stored at −80°C until use. For reverse transcription, 1 µg of total RNA was reverse transcribed using a First Strand cDNA Synthesis Kit (Thermo Scientific, K1622) according to the manufacturer’s protocol. Each reaction was prepared with a total volume of 20 µl and carried out at 37°C for 60 min in a Master Cycler Pro-S (Eppendorf). The resulting cDNA was considered optimally pure and used for reverse transcriptase-polymerase chain reaction (RT-PCR). PCR amplification was performed on 3 µl (<200 ng) of cDNA using HotStarTaq plus Master Mix (Qiagen) with Coral Load Concentrate (Qiagen, cat. no. 203443) Each PCR reaction was prepared with a total volume of 25 µl. The thermal cycler (Master Cycler Pro-S (Eppendorf)) was programmed according to the manufacturer’s instructions. After amplification, the samples were stored at 4°C in the short term or at −30°C to –15°C for longer storage. PCR products were confirmed by agarose gel electrophoresis. The gel was then visualized under a transilluminator (Vilber Lourmat). Primers are listed in Table 2.

To assess if mutations detected by NGS (please refer to “Results”) were also present in diagnostic bone marrow samples from the time of diagnosis, PCR primers flanking the identified mutations were designed (See Supplementary Table 1). The PCR contained 20–50 ng of genomic DNA, 1 x PyroMark PCR master mix (QIAGEN, Hilden, Germany), 1 x CoralLoad Concentrate (QIAGEN, Hilden, Germany), and 200 nM of each primer in a final volume of 25 μl. The PCR program was initiated with a denaturation step of 15 min at 95°C followed by 45 cycles of 20 sec at 95°C, 30 sec at 60°C, and 30 sec at 72°C. The final extension was performed for 10 min at 72°C. The PCR products were confirmed to be of the correct size by gel electrophoresis and subsequently Sanger sequenced in either forward or reverse direction by the service of Eurofins^®.