GSM2995357: P4-blood; Homo sapiens; OTHER
The characterized biopsies were subjected to sample isolation from the affected areas of the blocks using 1mm diameter disposable biopsy punches (Miltex). The QIAamp DNA FFPE Tissue kit (Qiagen) was used to extract DNA from FFPE tissues. The Qiagen AllPrep kit was used to extract both DNA and RNA from frozen O.C.T. embedded tissues. DNeasy Blood & Tissue kit (Qiagen) was used to extract DNA from blood samples. Library construction for exome sequencing was performed using Agilent Technologies SureSelect XT Reagent Kit, HSQ as described below. Briefly, 1-3 µg genomic DNA was sheared using Covaris S2 Focused-ultrasonicator, in a 130 µl volume, with 5.0 intensity, 10% duty cycle, 200 cycles per burst, and 6:00 minutes treatment time. Sheared DNA was purified using QIAquick PCR Purification kit (Qiagen). Using a combination of enzymes with filling and exonuclease activity, the DNA fragments became blunt ended. To prepare the fragments for accepting adaptors and to block concatamerization, an A overhang was added to the 3' end of the fragments, followed by ligation to adaptors with T overhangs. Adaptor-ligated fragments were amplified using 6 cycle PCR and followed by PCR product purification using Agencourt AMPure XP beads. Concentrations and fragment size ranges of the libraries were measured using NanoDrop spectrophotometer and D1K ScreenTape assay in the Agilent 2200 TapeStation system. Exome capture was performed on about 750 ng of the amplified libraries via biotinylated RNA bait molecules of SureSelect Human All Exon 50Mb (Agilent). The captured molecules were purified using Dynabeads MyOne Streptavidin T1, and index tagged in 10 PCR cycles. The amplified exome-enriched fragments were purified on Agencourt AMPure XP beads. Concentration and size measurements were done using Invitrogen Qubit dsDNA HS Assay and High Sensitivity D1K assay. The KapaBiosystems Kapa Library Quant kit was used to quantitate the concentration of adaptor ligated libraries. The captured libraries were sequenced using HiSeq 2000 101 cycle paired-end sequencing.
GEO Accession
GSM2995357