Blood cell culture dna mini kit

High molecular weight genomic DNA was isolated using Blood & Cell culture DNA Mini Kit (Qiagen) following the manufacturer protocol. Barcoded libraries were prepared by the SureSelect Human All Exon 50 Mb v4 kit (Agilent Technologies) and then sequenced on a SOLiD 5500xl sequencer (Life Technologies) with 50‐bp single fragment read. Raw reads were aligned onto the GRCh37 (hg19) reference genome with the SOLiDLifeScope software version 2.1. Variants and indels annotations were completed using an in‐house pipeline. Routine screening for recessive monogenic disease based on selection of variants with a frequency below 0.05% was performed. Synonymous variants, deep intronic variants, and variants in untranslated regions were excluded from the list of potentially causative genes.

DNA was extracted for 55 PDX samples using the Blood & Cell culture DNA mini kit (Qiagen) following the manufacturer's instructions. Illumina Infinium HumanCode‐24 BeadChip SNP arrays were used to analyze the DNA samples. Integragen SA (Evry, France) carried out hybridization, according to the manufacturer's recommendations. The BeadStudio software (Illumina) was used to normalize raw fluorescent signals and to obtain log R ratio (LRR) and B allele frequency (BAF) values. Asymmetry in BAF signals due to bias between the two dyes used in Illumina assays was corrected using the tQN normalization procedure (Staaf et al, 2008). We used the circular binary segmentation algorithm (Venkatraman & Olshen, 2007) to segment genomic profiles and assign corresponding smoothed values of log R ratio and B allele frequency. The Genome Alteration Print (GAP) method was used to determine the ploidy of each sample, the level of contamination with normal cells, and the allele‐specific copy number of each segment (Popova et al, 2009). Chromosomal instability index (CIN) was estimated by the mean number of SNP probes with a loss or gained status normalized by chromosomes length. SNP data are available through ArrayExpress (http://www.ebi.ac.uk/arrayexpress) under accession E‐MTAB‐5006.

The genomic DNA was isolated from cells using Blood & Cell Culture DNA Mini Kit (Qiagen, Germany) according to the manufacturer instructions. The PCR reactions were performed with 200 ng of DNA, Taq 2X Master Mix (New England Biolabs, MA), and a primer set specific for the Alu sequence (Supplementary Table 8). The PCR products were applied on 1.5% agarose gel electrophoresis and visualized with ethidium bromide (Sigma-Aldrich, MO). The 100 pb ladder (Takara Bio, Japan) was used as a size marker.
Total RNA was isolated with TRIzol (Thermo Fisher Scientific, MA), treated with DNase I (Promega, WI), and A260/A280 and A260/A230 ratios were measured with NanoDrop to assess RNA purity. Two μg of total RNA was reverse transcribed with GoScript™ Reverse Transcription System (Promega, WI) according to the manufacturer instructions and RT-qPCR reactions were conducted by Light Cycler 480 II using Light Cycler 480 SYBR Green I Master Mix (Roche Diagnostics, Germany). Gene expression levels were normalized to that of the beta actin (Actb) gene. The primer sets used for the PCR and RT-qPCR reactions are listed in (Supplementary Table 8).

Genomic DNA, extracted from breast cancer cells via a Blood & Cell Culture DNA Mini Kit (Qiagen, Germany), was used as a template to amplify the promoter of RP1. Briefly, the wild-type reporter fragment was amplified by PrimeStar HS DNA Polymerase with High GC Buffer (TaKaRa, Japan), digested by restriction enzymes Kpn I and Bgl II, ligated into the pGL4 vector digested by Kpn I and Bgl II, then sequenced; the correct clone was named pGL4-wt-RP1. A Site Directed Mutagenesis Kit (Clontech, Japan) was used to delete the KLF5 binding site in pGL4-wt-RP1 following the user instructions, and the correct clone after sequencing was named pGL4-del-RP1. All primers are listed in Table 1.
RP1, KLF5, p27kip1, and p300 overexpression and knock-down plasmids were all obtained from Genecoopia Technology (China).