title: GSM4504646: CLL_08_PreIbrutinib; Homo sapiens; OTHER
library_construction_protocol: For bulk tumor samples DNA was extracted from isolated CLL and AML cells using the Puregene Core kit A DNA extraction kit (# 158667, Qiagen, Maryland, USA) according to manufacturer recommendations for <1 million cells (Gentra Puregene Handbook).  DNA was quantified using spectrophotometric methods (Nanodrop 2000 from Thermo Scientific) using standard 260/280 OD ratios. 10ng of DNA from bulk samples are used for first step amplification. For low cell number samples (defined as <1000 sorted cells) DNA was prepared from sorted cells using the Single Cell Lysis Kit (#4458235, Invitrogen). Briefly, 9uL of Single Cell Lysis solution (no Dnase I) was directly added to sorted cell droplet. After incubating for 5 minutes at room temperature 1uL of Stop Solution was added to the reaction for 2 minutes at room temperature. 5uL of the total reaction is used for first step amplification. Libraries were prepared with Ion AmpliSeq Library kit2.0 (4475345) with a custom designed panel of AmpliSeq primers (CLL: IAD14429, 304 amplicons, 2 pools, 31.66kb panel size, 99.63% coverage; AML: IAD145356, 129 amplicons, 2 pools, 15.32kb panel size, coverage 100%; Supplementary Table 1, 2). To run multiple samples on the same chip we used IonExpress barcode adapters (kit#4471250 and #4474009). Libraries were constructed by first amplifying genomic DNA preparations with individual primer pools. Amplification was completed on the GeneAmp PCR system 9700 Dual 96-well thermal cycler from Applied Biosystems, using the following protocol: Initial Hold 99oC-2min followed by 21 cycles for the CLL panel (99oC-15sec, 60oC-4min), 20 cycles for the AML panel (99oC-15sec, 60oC-4min), and a final hold of 10oC. Corresponding PCR product pools were then combined and barcoded with IonExpress barcode adapters. Barcoded PCR products were purified with Agencourt AMPure XP kit (A63881 Beckman Coulter, Indianapolis, Indiana). While bulk extracted DNA libraries were eluted with low TE at this point and directly quantified, libraries constructed with <1000 cells underwent an additional amplification cycle to equalize library amounts using the following protocol: Initial hold of 98oC-2min followed by 5 cycles  (99oC-15sec, 64oC-1min) and a final hold of 10oC. All libraries were quantified using real time PCR with Ion Library TAQMAN Quantitation kit 44688022 on the Applied Biosystems ViiA7 Real Time PCR System instrument to allow for optimal final dilution of library for template preparation.
attributes.tag: GEO Accession
attributes.value: GSM4504646