Agarose gel cassettes

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).

MeD‐seq assays were essentially performed as previously described.
⁴³ (link) In short, 8 μL genomic DNA plasma‐derived cfDNA samples were digested with LpnPI (New England Biolabs, Ipswich, MA) yielding 32 bp fragments around the fully methylated recognition site containing a CpG. Samples were prepped for sequencing using the ThruPLEX DNA‐Seq 96D kit (Rubicon Genomics, Takara Bio Europe, Saint‐Germain‐en‐Laye, France) and purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science, Beverly, MA). Libraries were multiplexed and sequenced on an Illumina HiSeq 2500 for 50 bp single reads according to the manufacturer's instructions (Illumina, San Diego, CA).

MeD-seq assays were essentially performed as previously described [16 (link)]. In short, 8 µl genomic DNA (input ranged from 117 to 1728 ng) from frozen tissues, specified amounts of MCF7 genomic DNA or plasma-derived cfDNA were digested with LpnPI (New England Biolabs, Ipswich, MA) yielding 32 bp fragments around the fully methylated recognition site containing a CpG. Samples were prepped for sequencing using the ThruPLEX DNA-seq 96D kit (Rubicon Genomics, Takara Bio Europe, Saint-Germain-en-Laye, France) and purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science, Beverly, MA). Libraries were multiplexed and sequenced on an Illumina HiSeq 2500 for 50 bp single reads according to the manufacturer's instructions (Illumina, San Diego, CA). Samples were first sequenced until ~ 2 M reads and continued to a total of ~ 20 M reads only when the fraction of reads that passed the LpnPI filter (explained below) was at least 20%.