Qiaquick polymerase chain reaction purification kit

Synthetic genes (gBlocks) encoding the RNJ1 IgG HC, LC and scFv with 5′ T7 promoter and 3′ and 5′ untranslated regions (UTRs) were generated (Integrated DNA Technologies). The scFv gene was in frame with a C-terminal Flag tag for detection. The gBlocks were prepared following the manufacturer’s instructions and polymerase chain reaction amplified prior to IVT. The polymerase chain reaction products were purified using the QIAquick polymerase chain reaction Purification Kit (QIAGEN) and 0.5 µg underwent IVT using the HiScribe T7 Anti-Reverse Cap Analog mRNA Kit with tailing (New England Biolabs) according to the manufacturer’s instructions. The mRNA was purified using the Monarch RNA Clean-up Kit (New England Biolabs). A yield of ∼30 µg of mRNA was produced for each preparation.

The chromatin immunoprecipitation (ChIP) assay was performed as previously described (Sengupta et al., 2010 ; Obiorah et al., 2014 (link)). The antibodies used for the pull-downs were anti-ERα clone F-10X mouse monoclonal (2 μg/μl; 5 μg per reaction) (Santa Cruz Biotechnology), anti–steroid receptor coactivator 3 (SRC-3) clone AX15.3 mouse monoclonal (1 μg/μl; 5 μg per reaction) (Abcam, Cambridge, UK), and normal mouse IgG as intraperitoneal negative control (2 μg/μl; 5 μg per reaction) (Santa Cruz Biotechnology). The DNA fragments were purified using Qiaquick polymerase chain reaction (PCR) purification kit (Qiagen, Germantown, MD). Then, 2 μl of eluted DNA was used for RT-PCR analysis. The primer sequences used were GREB1 proximal ERE enhancer site amplification: 5′-GTG​GCA​ACT​GGG​TCA​TTC​TGA-3′ sense and 5′-CGA​CCC​ACA​GAA​ATG​AAA​AGG-3′ antisense (Integrated DNA Technologies). The data are expressed as percent input of starting chromatin material after subtracting the percent input pull-down of the intraperitoneal negative control.

Total RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) on the basis of the manufacturer’s instructions. After that, oligo (dT) magnetic beads were highly enriched with mRNA. Second-strand cDNA was then synthesized for 2 h at 16 °C using the total 20 µL first-strand product plus 80 µL of second strand mix containing 63 µL water, 10 µL second-strand buffer, 4 µL dNTPs, 2 µL DNA Polymerase and 1 µL of RNase H. Then, the cDNA fragments were purified with a QIAquick Polymerase Chain Reaction (PCR) Purification Kit (Qiagen, Hilden, Germany). The ligation products were selected by agarose gel electrophoresis, PCR amplified, as well as sequencing using Illumina HiSeq^TM 2000 (Illumina, Inc., San Diego, CA, USA).

The genome for this strain was obtained at the ultrasequencing core facility of the CRG, using Illumina GAIIx sequencing machine. DNA was fragmented by nebulization or in Covaris to a size approximately 300 bp. After shearing, the ends of DNA fragments were blunted with T4 DNA polymerase and Klenow fragment (New England Biolabs). Then, DNA was purified with a QIAquick polymerase chain reaction (PCR) purification kit (Qiagen). Thereafter, 3′-adenylation was performed by incubation with dATP and 3′–5′-exo-Klenow fragment (New England Biolabs). DNA was purified using MinElute spin columns (Qiagen), and double-stranded Illumina paired-end adapters were ligated to the DNA using rapid T4 DNA ligase (New England Biolabs). After another purification step, adapter-ligated fragments were enriched, and adapters were extended by selective amplification in an 18-cycle PCR reaction using Phusion DNA polymerase (Finnzymes). Libraries were quantified and loaded into Illumina flow cells at concentrations of 7–20 pM. Cluster generation was performed in an Illumina cluster station. Sequence runs of 2 × 76 cycles were performed on the sequencing instrument. Base calling was performed using Illumina pipeline software. In multiplexed libraries, we used 4-bp internal indices (5′-indexed sequences). Deconvolution was performed using the CASAVA software (Illumina).

The genotypic identification of the isolates was conducted by 16S rRNA gene sequencing technique. For amplification of the 16S rRNA gene, two universal primers, 27F (AGAGTTTGATCMTGGCTCAG) and 1492 R (TACGGYTACCTTGTTACGACTT) were used [65 ,66 (link)]. The PCR conditions were initial denaturation at 94 °C for 4 min, 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s and at 72 °C for 2 min, and a final extension at 72 °C for 5 min. The PCR products were purified using a Qiaquick Polymerase Chain Reaction (PCR) purification kit (Qiagen, Hilden, Germany). DNA sequence data sets were assembled using the Bioedit sequence alignment editor software, (version 7.0.9.0. Ibis Biosciences, Carlsbad, CA, USA) [67 ]. Sequence similarity values were identified using the basic local alignment search tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the National Centre for Biotechnology Information (NCBI, Rockville, MD, USA) and the accession numbers were obtained from GeneBank (NCBI, Rockville, MD, USA). Phylogenetic tree for nucleotides sequences was built up based on 16S rRNA gene sequences using Molecular Evolutionary Genetic Analysis software version 6 (MEGA 6; The Biodesign Institute, Tempe, AZ, USA), using the Neighbour-joining (NJ) method [68 (link)]. Sequence alignments were arranged by CLUSTAL W. A bootstrap was running out with 1000 replicates of the data.