TruePrime WGA of 1 cell of LOUCY cell line
Cell lysis and amplification was performed using the TruePrime Single cell WGA kit (Sygnis, Heidelberg, Germany), following manufacturers instructions. Samples were purified according to the manufacturers protocol of the Genomic DNA Clean & Concentrator kit (version 1.0.0, Zymo Research, Irvine, USA) with 5X binding buffer. Concentration was measured using Qubit dsDNA High Sensitivity Assay kit (Life Technologies, Carlsbad, USA). The sample quality was assessed with the Agilent 12000 (12K) DNA Assay kit (Bioanalyser, Agilent Technologies, California, USA). One hundred ng of the WGA product was fragmented to an average size distribution of 200?bp with the S2 Focused Ultrasonicator with Adaptive Focused Acoustics (AFA) technology (Covaris, Woburn, USA). Samples were diluted in 1/5X Tris-EDTA buffer (TE-buffer; stock 1X) to a volume of 130??l in microTUBES (Covaris, Woburn, USA). The programmed guidelines for fragmentation to 200?bp were followed (Duty cycle of 10%, Intensity of 5 and 200 cycles/burst), but the fragmentation time was prolonged to 190s based on previous experience. Subsequently, libraries were created using NEBNext Ultra DNA Library Prep (PCR 1.4A, New England Biolabs, Ipswich, USA), following manufacturers protocol, with the exception of some minor modifications. After incubation with the USER enzyme, a DNA purification step (Zymo Genomic DNA Clean & Concentrator) was included before the size selection step. Size selection was performed with the E-Gel iBase Power system (Invitrogen) using an E-gel EX 2% agarose gel and a 1?kb Plus DNA ladder (Thermo Fisher Scientific, Waltham, USA). For all samples, fragments with a size of ~300?bp were cut from the gel, and DNA was recovered using the Zymoclean gel DNA recovery kit (Zymo Research). The size selected DNA samples were then subjected to an enrichment PCR using NEBNext Multiplex Oligos for Illumina (Index Primers Set 1 and 2 version 3.0) according to the protocol. tRNA was added to the reaction to minimize the loss of DNA via tube interaction. The quality of the different libraries was assessed with the Agilent High-Sensitivity DNA kit (Bioanalyser, Agilent Technologies, California, USA). One hundred ng of the WGA product was fragmented to an average size distribution of 200?bp with the S2 Focused Ultrasonicator with Adaptive Focused Acoustics (AFA) technology (Covaris, Woburn, USA). All samples were diluted in 1/5X Tris-EDTA buffer (TE-buffer; stock 1X) to a volume of 130??l in microTUBES (Covaris, Woburn, USA). The programmed guidelines for fragmentation to 200?bp were followed (Duty cycle of 10%, Intensity of 5 and 200 cycles/burst), but the fragmentation time was prolonged to 190s based on previous experience. The PCR-free libraries were created entirely according to the TruSeq DNA PCR-free HT sample preparation kit (Illumina), which does not require an enrichment PCR. For each sample, 1??g of WGA product was used as input. From here onwards, these samples will be referred to as PCR-free samples. Before sequencing the samples, a library quantification was performed using a Sequencing Library qPCR Quantification kit (Illumina, San Diego, USA) to quantify the sequenceable DNA fragments containing the correct adapters. The control template used for the standard curve was a PhiX control library (10?nM). The libraries from the different samples were equimolarly pooled, denatured and diluted to a final loading concentration of 2.1?pM for sequencing. Finally, single-end index 75?bp sequencing was performed on a high-output flowcell on a NextSeq500 (Illumina, California, USA).