Zr rna microprep kit

The ZR RNA MicroPrep kit (Zymo Research Corporation, CA, USA) was used for miR extraction from 30 μl microdialysate or 80 μl EDTA plasma and eluted in 6μl nuclease-free water. A spike in control, Caenorhabditis elegans miR-67-3p (Life technologies, CA, USA), was added prior extraction.

RNA from 20 to 40 imaginal discs was extracted with ZR-RNA MicroPrep Kit from Zymo Research following the manufacturer’s instructions. Sequencing libraries were prepared using TruSeq Stranded mRNA Library Preparation Kit from Illumina and following the manufacturer’s instructions. Sequencing was performed in a HiSeq sequencer from Illumina at the Ultrasequencing Unit of the Centre for Genomic Regulation (CRG, Barcelona, Spain). A minimum of 50 million paired-end 75 bp-long reads were obtained per replicate and two replicates were performed per each tissue.

Total RNA was prepared from the EBV-B cells of individuals heterozygous for IRF4 mutations (two patients and two asymptomatic relatives), and healthy homozygous WT relatives (n = 4). We also included samples from unrelated individuals (seven healthy controls and 25 patients with Tw carriage; all IRF4 coding exons for each individual were sequenced and shown to be WT). Moreover, the 25 WD patients included in this experiment were found to have an intact IRF4 cDNA structure and normal levels of IRF4 protein production in EBV-B cells. RNA was prepared from 500,000 cells with the ZR RNA Microprep kit (Zymo Research), according to the manufacturer’s instructions. A mixture of random octamers and oligo dT-16 was used, with the MuLV reverse transcriptase (High-Capacity RNA-to-cDNA kit, Thermo Fisher Scientific), to generate cDNA. Quantitative real-time PCR was performed with the TaqMan Universal PCR Master Mix (Thermo Fisher Scientific), the IRF4-specific primer (Hs01056533_m1, Thermo Fisher Scientific) and the endogenous human β-glucuronidase (GUSB) as a control (4326320E, Thermo Fisher Scientific). Data were analyzed by the ΔΔCt method, with normalization against GUSB.

RNA from prophase I meiocytes from domesticated and wild genotypes was isolated using the ZR RNA MicroPrep kit (Zymo Research, Orange, CA) following the manufacturer’s instructions and modifications for sRNAs extractions, and stored at −70 °C. Two libraries (one for each genotype) were prepared using standard Illumina TruSeq Small RNA library preparation kits, and sequenced using the Illumina HiSeq 2500 platform to obtain 37 bp single-end reads. Reads were quality-trimmed using the Kraken set of tools [88 (link)], and those reads with a length between 20 and 25 nt were selected for subsequent analyses. Mapping of the sRNA reads was performed using the Bowtie 1.1.2 version [89 (link)] to a mixed reference of the genome draft contigs (described above) and meiocyte transcriptome assembly from this study with the following parameters: −v 1 --best --strata -a -f --chunkmbs 512.