Paired end dna sample prep kit

The whole genome of strain TBCP-5362 was sequenced using a PacBio RS II (Pacific Biosciences, Menlo Park, CA, United States), with a depth of approximately 100-fold coverage. The reads produced with the PacBio RS II were de novo assembled using MaSuRCA (Tallon et al., 2014 (link); Kuang et al., 2015 ). The other 30 strains were sequenced using Illumina Paired-End sequencing technology (Illumina, Little Chesterford, Essex, United Kingdom), with a depth of 90–100-fold coverage. A library for Illumina Paired-End sequencing was prepared from 5 μg DNA using a Paired-End DNA Sample Prep Kit (Pe-102-1001, Illumina Inc., Cambridge, United Kingdom). Libraries prepared using Nextera technology and paired end reads of either 100 bp (Illumina HiSeq 2000). DNA was fragmented by nebulization for 6 min at a pressure of 32 psi. For end-repair and phosphorylation, sheared DNA was purified using the QIAquick Nucleotide Removal Kit (Qiagen, Crawley, United Kingdom). The end-repaired DNA was A-tailed and adaptors were ligated according to the manufacturer’s instructions. De novo assembly was performed using Velvet Optimiser v2.2 (Zerbino and Birney, 2008 (link)). Genome sequence annotation was conducted using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline¹. In addition, 98 publicly available Citrobacter genomes were obtained from NCBI GenBank.

Paired-end sequencing libraries with an insert size of about 500 bp were constructed using the Paired-End DNA Sample Prep Kit (Illumina Inc., San Diego, CA, United States). The libraries were sequenced on an Illumina HiSeq 2500 system using standard protocol (Illumina, Inc.; San Diego, CA, United States). The sequence depth of two pools was 30× on average.
Raw reads were processed to obtain high-quality clean reads according to three stringent filtering standards: 1) removing reads with ≥10% unidentified nucleotides (N); 2) removing reads with >50% bases having Phred quality scores of ≤20; and 3) removing reads aligned to the barcode adapter.

The construction of DNA libraries was performed using a Paired-End DNA Sample Prep Kit (Illumina, CA, USA) and a Multiplexing Sample Preparation Oligonucleotide Kit (Illumina) according to the manufacturer’s instructions. In this step, ∼350-bp fragments were collected from each sample using E-Gel SizeSelect 2% (Invitrogen). The constructed libraries were subsequently subjected to deep sequencing using a Genome Analyzer II (Illumina). These reads were mapped to the rice genome with the Burrows-Wheeler Aligner (BWA) software package [31] (link) using the International Rice Genome Sequencing Project genome sequence (build 5) from the Rice Annotation Project Database (RAP-DB: http://rapdb.dna.affrc.go.jp) as the reference genome sequence. The OsTGAP1-binding regions in each sample were then detected using Partek Genomics Suite (ver. 6.5; http://www.partek.com/, Partek Software, MO, USA) according to the following thresholds: window size = 100, peak cut-off false discovery rate (FDR) <0.001, strand separation FDR<0.05, and significant enrichment in ChIP DNA compared to the ‘Input’ control (FDR<0.05). The sequence data were deposited in the DDBJ Sequence Read Archive (http://trace.ddbj.nig.ac.jp/dra/index.html; ID: DRA001274).

The genomic library was prepared using the Paired-End DNA Sample Prep Kit (Illumina, San Diego, CA USA) following the manufacturer’s protocol. Genomic DNA was sequenced on the Illumina Genome Analyzer IIx (GAIIx, 2×150 bp reads); the paired-end library insert size averaged 430 bp. Total RNA was converted into a cDNA fragment library with an average insert size of 354 bp using Illumina’s mRNA-Seq kit (San Diego, CA USA), according to the manufacturer’s protocol; paired-end 2×100 bp reads were also sequenced on the Illumina GAIIx sequencer. Quality trimming and adaptor removal for both DNA and RNAseq reads were done with FASTX-Toolkit (Version 0.6.1). Genomic DNA trimmed reads were assembled with the CLC Genomics Workbench (Aarhus, Denmark). Scaffolding of the assembled contigs was done using SSPACE [91 (link)]. cDNA sequences were assembled into ESTs using the CLC Genomics Workbench and ABySS using three different k-mer values (k = 56, 63, 70) [92 (link),93 (link)]. Merging the four transcriptome assemblies and removing short redundant contigs was done using BLAT/ CD-HIT-EST [94 (link),95 (link)].

Briefly, 2 × 10⁸ breast cancer cells were collected from eight individual samples. The cells were fixed for 10 min at room temperature with 1% formaldehyde-containing medium and sonicated ten times for 5 s each. The sonicated chromatin samples were incubated at 4 °C overnight, with the anti-FOXP3 antibody. From each ChIP reaction mixture, 10% was kept as input DNA. Pre-rinsed protein A/G plus agarose beads (final concentration 75 ng/μl) were added to each ChIP reaction mixture and incubated for 60 min at room temperature. The beads were then incubated in 100 μl of elution buffer at 65 °C for 20 min to elute the immunoprecipitated DNA. ChIP-Seq libraries were prepared, using a Paired-End DNA Sample Prep kit (Illumina, PE-102-1001), and were run on HiSeq 4000 Sequencing Systems (Illumina). All sequencing data were mapped to the February 2009 human genome assembly (GRCh37/hg19), and peak calling was performed, using the Model-based Analysis of ChIP-Seq (MACS) version 2.1.1 (http://liulab.dfci.harvard.edu/MACS) with the default parameters to obtain the primary binding regions. For ChIP-Seq analysis, the background was subtracted, and the primary binding regions were filtered based on 5–50-fold enrichment and q value < 0.01.