GSM1342799: 1306734 Tumor [methylation]; Homo sapiens; Bisulfite-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Bisulfite-Seq
Antigen
Bisulfite-Seq
Cell type
Cell type Class
Liver
Cell type
Hepatocellular carcinoma
NA
NA
Attributes by original data submitter
Sample
source_name
liver, HCC tumor
disease status
hepatocellular carcinoma (HCC)
tissue
liver tumor
gender
male
patient
1306734
Sequenced DNA Library
library_strategy
Bisulfite-Seq
library_source
GENOMIC
library_selection
RANDOM
library_construction_protocol
Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen). 1µg of genomic DNA was fragmented into approximately 200-300bp using a Covarias sonication system (Covarias). After purification, the fragmented fractions were treated with a mix of T4 DNA polymerase, Klenow Fragment and T4 polynucleotide kinase to repair blunt and phosphorylate ends. The blunt DNA fragments were subsequently 3' adenylated using Klenow Fragment (3'-5' exo-) and then ligated by T4 DNA Ligase to adapters that were synthesized with 5'-methylcytosine instead of 5'-cytosine and contain index sequences inside. After each step, the reaction products were purified using QIAquick PCR purification kit (Qiagen). The constructed libraries were quantified using a Qubit fluorometer with Quant-iT dsDNA HS Assay Kit (Invitrogen). The liquid hybridization capture procedure was then performed as follows. Briefly, 200 ng DNA from each of five adapter-ligated libraries were pooled together. 10 µg human Cot-1 DNA and 1nmol of adapter complementary DNA oligos were added and subsequently were dried using a SpeedVac (eppendorf) at 60°C. After that, the mixture was denatured at 95°C for 10min in a final reaction volume of 10.5µl consisting of 7.5µl 2X SC Hybridization Buffer and 3µl SC Hybridization Component A. After centrifugation, 4.5µl of designed probes were added and the mixture was hybridized in a thermal cycler at 47°C for 72 hours with the lid heated at 57°C. After hybridization, the biotinylated probes bounded with the target DNA were captured using 100µl of Dynabeads® M-280 Streptavidin (Invitrogen), which had been pre-washed two times with a total of 400µl Streptavidin Dynabead Binging and Washing Buffer. The capture program was 47°C for 45min in a thermal cycler with the lid heated at 57°C and vortexing for 3 seconds at 15min intervals. Discarded the unbounded fractions and washed the collected DNA-probe-beads complex with 100µl of 47°C pre-warmed 1X Wash Buffer I for one time and with a total of 400µl of 47°C pre-warmed 1X Stringent Wash Buffer I for two times with the incubations of 47°C for 5min. After discarding the supernatant, the collected beads complex was again washed with 200µl of 1X Wash Buffer I, 1X Wash Buffer II and 1X Wash Buffer III respectively. Finally, the captured DNA was eluted in 50µl of 10M NaOH with incubation at room temperature for 10min. The supernatant was transferred into a new tube and neutralized with 50µl of 10M HAc and then purified using MiniElute PCR purification Kit (Qiagen). For bisulfite conversion, 200ng unmethylated lambda DNA was added into each captured product as carriers and ZYMO EZ DNA Methylation-Gold Kit™ (ZYMO) was employed to convert unmethylated cytosine into uracil according to the instructions. After purification, PCR was carried out in a final reaction volume of 50µl consisting of 20µl converted products, 4µl 2.5mM dNTP, 5µl 10×buffer, 0.5µl JumpStart™ Taq DNA Polymerase (SIGMA), 1µl PCR primers1.0, 1µl PCR index primers (which were used to identify different captures) and 18.5µl water. The following thermal cycling program was 94°C 1 min, 15 cycles of 94 °C 10s, 58°C 30s, 72°C 30s then prolong at 72°C for 5min and hold at 12°C. The PCR products were purified using AMPure beads (Agencourt), quantified by the Bioanalyzer analysis system (Agilent) and real time PCR assay and then analyzed using Illumina Hiseq 2000. All of these samples were pooled into 7 libraries with a one-to-one correspondence 7bp barcode. The barcode information is following: s02_C-AATAACT-pooling1, s14_P-TGTAATT-pooling1, s96_P-ACTTGAT-pooling1, s00_C-AATAACT-pooling2, s13_C-TGTAATT-pooling2, s95_C-ACTTGAT-pooling2, s09_C-ATAATCT-pooling3, s15_P-TGTAATT-pooling3, s06_P-GTACATT-pooling4, s10_P-TATGCTT-pooling4, s16_C-TGTAATT-pooling4, s01_P-GTACATT-pooling5, s99_P-CGAATAT-pooling5, s95_C-CGAATAT-pooling6, s96_P-ACTTGAT-pooling6, s05_C-GTACATT-pooling7, s11_P-TATGCTT-pooling7, s12_C-TGTAATT-pooling7. The constructed libraries were quantified using a Qubit fluorometer with Quant-iT dsDNA HS Assay Kit (Invitrogen).