GSM5033550: AML_test_A78; Homo sapiens; Bisulfite-Seq
Diagnostic DNA of AML patients was either isolated from whole peripheral blood with MagCore system (RBSBioscience) or from peripheral blood leukocytes lysed in RLT buffer using AllPrep DNA/RNA Mini Kit (Qiagen). Healthy donors' DNA was isolated with MagCore system from CD34+ cells harvested from buffy coats using MicroBeads kit (Miltenyi Biotec). Each sequencing library consisted of 16-18 samples and libraries were prepared according to SeqCap Epi protocol (Roche) with KAPA HyperPrep Kit (Roche). First, Bisulfite-conversion Control (unmethylated DNA from Enterobacteria phage lambda, a component of SeqCap Epi Accessory kit (Roche)) was added to 800–1,200 ng of patients/donors' DNA. This DNA mixture was subsequently fragmented either via Bioruptor Pico instrument (Diagenode) or E220 Focused ultrasonicator (Covaris) to get ~ 200 bp DNA fragments. The size of fragments was checked in two random samples from each library on 4200 TapeStation System (Agilent Technologies) using Agilent D1000 ScreenTape (Agilent Technologies). EZ DNA Methylation Lightning Kit (Zymo Research) was used for the bisulfite conversion, as recommended in the SeqCap Epi workflow. Each library was hybridized for ~ 68 hours with probes from our custom DNA methylation sequencing panel (made by Roche). KAPA Library Quantification Kit (Roche) was utilized to assess the final concentration of sequencing libraries. Average size of the libraries' fragments was measured on 4200 TapeStation System with Agilent D1000 ScreenTape (Agilent Technologies). Sequencing was performed using MiSeq Reagent Kit v2 (300-cycles) (Illumina) on MiSeq instrument (Illumina). targeted bisulfite sequencing using custom probes
GEO Accession
GSM5033550