Thruplex dna seq 96d kit

12.3 to 96.1 ng of gDNA, quantified with Qubit dsDNA BR Assay kit (Thermo Fisher Scientific), was fragmented with Episonic Multi-Functional Bioprocessor 1100 (Epigentek Group Inc., Farmingdale, NY, USA). The libraries were created with Thruplex DNA-seq 96D kit (Rubicon Genomics) according to manufacturer’s protocol. 10–50 ng of DNA was used for library preparation. Five amplification cycles were used. The amplified libraries were purified with 50 µl of NucleoMag NDS Clean-up and Size Select beads and eluted to 30 µl of PCR grade water. The libraries were quantified with LabChip GX HT DNA HiSens chip (PerkinElmer, Waltham, MA, USA) (Supplementary Data 2).

MeD-seq assays were performed as previously described [20 (link)]. Briefly, 20 µL genomic DNA (input 90 ng) from frozen liver tissues was digested with LpnPI (New England Biolabs), generating 32 bp fragments around the methylated recognition site containing a CpG. These short DNA fragments were further processed using the ThruPlex DNA–seq 96D kit (Rubicon Genomics, Ann Arbor, MI, USA). Stem–loop adapters were blunt-end ligated to repaired input DNA, then amplified to include dual-indexed barcodes using a high-fidelity polymerase to generate an indexed Illumina NGS library. The amplified end product was purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science, Beverly, MA, USA). Multiplexed samples were sequenced on Illumina NextSeq2000 systems for paired-end reads of 50 bp, according to the manufacturer’s instructions. The dual-indexed samples were de-multiplexed using bcl2fastq v2.20 software (Illumina, San Diego, CA, USA).

MeD-seq analyses were essentially carried out as previously described [22 (link)]. In brief, 22 DNA samples were digested by LpnPI (New England Biolabs, Ipswich, MA, USA), resulting in snippets of 32 bp around a fully methylated recognition site that contains a CpG. These short DNA fragments were further processed using the ThruPlex DNA–seq 96D kit (Rubicon Genomics Ann Arbor, MI, USA). Stem-loop adapters were blunt-end ligated to repair input DNA and amplified to include dual indexed barcodes using a high-fidelity polymerase to generate an indexed Illumina NGS library. The amplified end product was purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science, Beverly, MA, USA). Multiplexed samples were sequenced on Illumina HiSeq2500 systems for single read of 50 bp according to manufacturer’s instructions. Dual indexed samples were de-multiplexed using bcl2fastq software (Illumina, San Diego, CA, USA).

Methylated DNA sequencing (MeD-seq) was used to analyze genome-wide DNA methylation. MeD-seq provides single-nucleotide resolution by exploiting the properties of the DNA methylation dependent restriction enzyme LpnPI (27 (link)). This enzyme generates DNA fragments of 32 base pairs (bp) by cutting 16 bp downstream from the methylated CpG sites, which allows specific focus on the methylated regions. The MeD-seq analyses were essentially carried out as previously described (27 (link), 28 (link)). In brief, DNA samples were digested by LpnPI (New England Biolabs, Ipswich, MA, USA), resulting in snippets of 32 bp around a fully-methylated recognition site that contains a CpG. These short DNA fragments were further processed using a ThruPlex DNA-seq 96D kit (cat no. R400407, Rubicon Genomics Ann Arbor, MI, USA) and a Pippin system. Stem-loop adapters were blunt-end ligated to repaired input DNA and amplified to include dual indexed barcodes using a high-fidelity polymerase to generate an indexed Illumina NGS library. The amplified end product was purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science, Beverly, MA, USA). Multiplexed samples were sequenced on Illumina HiSeq2500 systems for single read of 50 bp according to the manufacturer’s instructions. Dual indexed samples were demultiplexed using bcl2fastq software (Illumina, San Diego, CA, USA).

DNA from iPSC samples collected at passage 12 were used for MeD‐seq analysis. LpnPI and MspJI (New England Biolabs) digestions were carried out according to the manufacturer's protocol. Reactions contained 50 ng in a 10‐μL volume and digestion took place overnight in the absence of enzyme activators. Digests of genomic DNA with LpnPI resulted in snippets of 32 bp around the fully methylated recognition site that contains CpG. The DNA concentration was determined by the Quant‐iT High‐Sensitivity assay (Life Technologies; Q33120) and 50 ng ds DNA was prepared using the ThruPlex DNA‐seq 96D kit (Rubicon Genomics cat#R400407). Twenty microliters of amplified end product was purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science). Stem‐loop adapters were blunt‐end ligated to repaired input DNA and amplified (4 + 10 cycles) to include dual‐indexed barcodes using a high fidelity polymerase to yield an indexed Illumina NGS library. Multiplexed samples were sequenced on Illumina HiSeq2500 systems for single read of 50 base pairs according to the manufacturer's instructions. Dual‐indexed samples were demultiplexed using the bcl2fastq software (Illumina).