Truseq chip sample preparation kit

NIPTmer was tested using two sets of cfDNA samples of pregnant women, one from Estonia and another from Belgium. The Estonian sample set consisted of 294 samples and was used to train NIPTmer algorithm. Sequencing libraries were generated using Illumina TruSeq ChIP Sample Preparation kit (Illumina Inc., San Diego, CA, USA) and sequenced on Illumina NextSeq 500 instrument with an average coverage of 0.32 × (minimum 0.08, maximum 0.42) producing 85 bp single-end reads. The Belgian dataset, published previously^{3 (link)}, consisted of 289 samples and was used in a blind test to validate the algorithm by comparing the results of the two pipelines. Libraries were prepared using TruSeq ChIP Sample Preparation kit (Illumina) and sequenced on Illumina HiSeq 2500 producing 50 bp single-end reads with an average coverage of 0.13x^{3 (link)}.
The study was approved by Research Ethics Committee of the University of Tartu (#246/T-21) and written informed consent was obtained from all participants. All experiments were performed in accordance with relevant European guidelines and regulations.

For ChIP-seq 10⁸ asynchronously growing cells were crosslinked with 1 % formaldehyde for 10 min at room temperature, followed by quenching with 125 mM glycine for 10 min, washed twice with 1× phosphate buffered saline (PBS), and resuspended in chromatin immunoprecipitation (ChIP) lysis buffer (150 mM NaCl, 1 % Triton X‐100, 0.1 % SDS, 20 mM Tris–HCl pH8.0, 2 mM EDTA). Chromatin was sheared to an average length of 200–500 bp using a Bioruptor sonicator. After overnight incubation with DiaMag magnetic beads (Diagenode, Inc.) and CTCF or BORIS monoclonal or polyclonal antibodies (characterized and described by us [29 (link), 32 (link), 39 (link)]), precipitated chromatin was then washed, de-crosslinked, and digested with proteinase K. The resulting DNA was purified using phenol/chloroform/isoamyl alcohol. DNA concentration was assessed with a Quant‐it PicoGreen dsDNA kit (Invitrogen) and 5–10 ng was used to generate sequencing libraries. ChIP DNA was amplified using a TruSeq ChIP Sample Preparation Kit (Illumina, Inc., USA). Briefly, the immunoprecipitated material was end-repaired, A-tailed, ligated to the sequencing adapters, amplified by 15 cycles of PCR, and size selected (200–400 bp) followed by single end sequencing on an Illumina Genome Analyzer according to the manufacturer’s recommendations.