Rna 6000 pico chip

Total RNA was extracted from kidney tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For the quality assessment and quantification of RNA, Agilent 2100 bioanalyzer using the RNA 6000 Pico Chip (Agilent Technologies, Amstelveen, The Netherlands) and NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA) were used, respectively. The miRNA library was constructed using NEBNext Multiplex SmallRNA Library Prep kit (New England BioLabs, Inc., USA) according to the manufacturer’s protocol.
Sequence reads were mapped in order to obtain bam file by bowtie2 software tool, using mature miRNA sequence as a reference. Read counts mapped on mature miR-seq were extracted from the alignment file using bedtools v2.25.0 [30] (link) and Bioconductor [31] (link) using R statistical programming language [32] (link). It was also utilized to determine the amount of expression of miRNAs. For comparison between samples, the normalization method of counts per million (CPM) and trimmed mean of M-values (TMM) was used.

Rumen liquor sampled at 4 h post feeding on two consecutive days (~1 ml each day) was mixed with RNA later and stored for further use at –80°C. Total RNA was extracted from each sample and purified by RNeasy Mini Kit (Qiagen). Removal of ribosomal RNA was done by Ribo-Zero rRNA Removal Kit (Epicentre). Quality and quantity of mRNA was assessed on an RNA 6000 Pico Chip on the Agilent 2100 Bioanalyzer instrument. Total mRNA was fragmented and barcoded cDNA libraries were prepared for GS-FLx titanium platform according to manufacturer’s protocol. The emulsion-based clonal amplification (emPCR amplification) of cDNA libraries was carried out followed by sequencing by GS FLX Titanium (Roche, USA).

Frozen tissue samples (70–190 mg) were homogenized in 2-ml TRIzol^® Reagent (Ambion) using a Ultra-Torrax T25 homogenizator (Labortechnik). Total RNA was extracted RiboPure Kit (Ambion) according to manufacturers´ instructions. Poly(A) RNA was enriched from 1-μg total RNA using MicroPoly(A)Purist Kit (Ambion) according to the manufacturer’s instructions. The quantity and quality of the input RNA were controlled using a RNA 6000 Pico chip on a Bioanalyzer (Agilent Technologies), and only RIN values above 7 were used in the analysis.
cDNA library preparation was conducted at the Uppsala Genome Centre (SciLifeLab). Briefly, an rRNA depletion step was performed with 56 mg as input amount for all samples, using the RiboMinus Eukaryote Kit (Life Technologies). Whole-transcriptome libraries were then constructed using the SOLiD Total RNA-Seq Kit (rev B, July 2011, Life Technologies). Emulsion PCR was performed using the SOLiD EZ Bead System (Life Technologies), and the libraries were then sequenced on three lanes with the SOLiD 5500xl System (Life Technologies).

Total RNA samples were sent to the Massively Parallel Sequencing Shared Resource (MPSSR) at Oregon Health and Science University (OHSU) for library preparation. Briefly, total RNA concentration and sample integrity was assessed using an Agilent RNA 6000 Pico chip on an Agilent Technologies 2100 Bioanalyzer instrument (Figure 4—source data 1). Following quantification and quality control, 325 ng of total RNA was subjected to ribosomal RNA reduction via Epicenter’s Ribo-Zero Gold kit (Human/Mouse/Rat). The output was then used with Illumina’s TruSeq RNA Sample Preparation Kit v2, beginning at the RNA fragmentation step. Poly(A) selection was not performed due to limited starting material. Barcode indexing adapters were ligated and all 10 samples (5 control and five injured) were sequenced (100 bp single end reads) on a single flow cell lane on the Illumina HiSeq 2500 Seqeuncer. Across samples, an average of 85.2% of the fragments mapped to the Drosophila genome; 41.2% of these mapped to exons and 9% to introns (Figure 4—source data 1). Notably, 22% of reads corresponded to ribosomal RNA (rRNA) and small nuclear RNA (snRNA). Enhanced ribosomal RNA mapping and reduced exon mapping may have been influenced by the fact that our ribosomal RNA depletion kit was not specific to Drosophila.

Ribosomal footprints were isolated with a sucrose cushion, size-selected, and dephosphorylated as previously described [2 (link), 11 (link)]. Following dephosphorylation of size-selected footprints, we determined the concentration of input material using a Bioanalyzer (RNA 6000 Pico Chip, Agilent Technologies). We found that quantification with a Bioanalyzer was more accurate than with a RNA Qubit or Nanodrop due to the presence of Glycoblue (Ambion) as a precipitant. We used a newly developed kit for small RNA library construction (SMARTer® smRNA-Seq Kit for Illumina®, Clontech catalog number 635030) to generate ligation-free ribosome profiling libraries. Between 1 and 5 ng of size-selected material was used as input and diluted with water to a total volume of 7 μL. Ensuring that reagents remained on ice, polyadenylation mix was prepared by combining 7 μL of RNA input with 2.5 μL of mix 1, which includes poly(A) polymerase. After adding the polyadenylation mix, samples were incubated for 5 minutes at 16 °C. Following incubation, samples were immediately placed on ice to ensure the poly(A) tailing reaction did not continue.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen) with on-column DNase digest. The concentration and quality of extracted RNA were evaluated using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific) and the RNA 6000 Pico Chip (Agilent Technologies), respectively. Subsequently, 500 ng of RNA was used to perform an Illumina sequencing library preparation using the QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen) according to the manufacturer's protocol. During library preparation 15 PCR cycles were used. Libraries were quantified by qRT-PCR, according to Illumina's protocol 'Sequencing Library qPCR Quantification protocol guide', version February 2011. The library’s size distribution and quality were analysed using the High sensitivity DNA Chip (Agilent Technologies) on a 2100 Bioanalyzer platform (Agilent Technologies). Sequencing was performed on a NextSeq 500 (Illumina), generating 75 bp single-end reads. Two separate sequencing runs where performed, one for the undifferentiated hESC samples and one for the 24-h ME differentiation samples. All data were deposited in the GEO repository with accession number GSE148050.
Details on the downstream bioinformatic analysis of mRNA sequencing can be found in the Supplementary Information.