RNA from bead-bound PCM-1(+) nuclei was collected by using the RNEasy Plus Micro kit (Qiagen, Hilden, Germany). Nuclear RNA sequencing (RNA-seq) libraries were constructed by using the Stranded RNA-seq Kit with Ribo Erase (Kapa Biosystems Inc.) with custom Y-shaped adapters. Paired-end 2×75 bp sequencing was performed for RNA-seq libraries with an Illumina Nextseq instrument (DNA Link). Reads were first mapped to the mouse genome (mm10) by using STAR (Dobin et al., 2013 ). Differential expression analysis was then carried out with DESeq2 (Love et al., 2014 (link)). Gene ontology analysis was performed by using Metascape [http://metascape.org ] (Tripathi et al., 2015 (link)), and displayed using GOplot (Walter et al., 2015 ). Gene set enrichment analysis using publicly available data(Uosaki et al., 2015 (link)) was performed by interrogating the top 200 most enriched transcripts in either adult hearts relative to embryonic (E12-14); or embryonic relative to adult against our RNA-seq dataset (enrichment score is relative to control) (Mootha et al., 2003 (link); Subramanian et al., 2005 (link); Uosaki et al., 2015 (link)).
Riboerase
Manufactured by Roche
Sourced in Germany, United States
RiboErase is a laboratory equipment product designed for the removal of ribosomal RNA (rRNA) from RNA samples. It enables the selective depletion of rRNA, which is essential for various downstream RNA analysis applications, such as RNA sequencing and transcriptome profiling.
Automatically generated - may contain errors
Lab products found in correlation
Kapa rna hyperprep kit, by Roche (7 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Hiseq 2500, by Illumina (4 mentions)
Kapa stranded rna seq kit, by Roche (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Hiseq 3000, by Illumina (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
Hiseq 4000, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Ribo zero, by Illumina (2 mentions)
On column dnase digestion, by Qiagen (2 mentions)
Kapa rna hyperprep, by Roche (2 mentions)
Stranded rna seq kit, by Roche (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Truseq index adapters, by Illumina (1 mentions)
2100 bioanalyzer, by Illumina (1 mentions)
Rnaeasy extraction kit, by Qiagen (1 mentions)
Qubit 2.0 fluorometer, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
24 protocols using riboerase
1
Cardiac transcriptome profiling by nuclear RNA-seq
2
RNA Extraction and Library Preparation for Hepatic Transcriptome Sequencing
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Total RNA was extracted from hepatic tissue using an RNAeasy extraction kit from QIAGEN®30 with a DNAse cleaning step according to the manufacturer’s instructions. RNA quality was measured using Nanodrop, Qubit 2.0 fluorometer and Bioanalyzer 2100 instruments.
Fifteen libraries (one per individual) were constructed from 1.0 µl of total RNA using the KAPA Stranded RNA-Seq kit with RiboErase from KapaBiosystems®. Ribosomal RNA was removed by depletion by DNA primer hybridization followed by treatment with RNAse and DNAse according to the manufacturer’s instructions. The fragmentation cycle was adjusted to 10 cycles of amplification at 94 °C for 5 minutes to obtain fragments between 100 and 2100 bp in length. Each library was enriched with Illumina® TruSeq Index Adapters (250 nM) for multiplex sequencing.
Library quality was evaluated using a Qubit 2.0 fluorometer and an Agilent Bioanalyzer 2100; good quality libraries were paired-end (PE) sequenced on an Illumina HiSeq. 4000 at the Center for Genomics Services of the University of California, Berkeley.
Fifteen libraries (one per individual) were constructed from 1.0 µl of total RNA using the KAPA Stranded RNA-Seq kit with RiboErase from KapaBiosystems®. Ribosomal RNA was removed by depletion by DNA primer hybridization followed by treatment with RNAse and DNAse according to the manufacturer’s instructions. The fragmentation cycle was adjusted to 10 cycles of amplification at 94 °C for 5 minutes to obtain fragments between 100 and 2100 bp in length. Each library was enriched with Illumina® TruSeq Index Adapters (250 nM) for multiplex sequencing.
Library quality was evaluated using a Qubit 2.0 fluorometer and an Agilent Bioanalyzer 2100; good quality libraries were paired-end (PE) sequenced on an Illumina HiSeq. 4000 at the Center for Genomics Services of the University of California, Berkeley.
3
RNA-seq of B cells and U2OS cells
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Total RNA was isolated using Trizol reagents (15596018; Life Technologies) and was treated with DNase I (M6101; Promega). RNA-seq libraries were prepared using KAPA stranded RNA-Seq kits with RiboErase (HMR) (KR1142; KAPA) following the manufacturer’s instructions. Then these libraries were sequenced with Illumina HiSeq at Geneseeq company. About ~25 million of 2 × 150 bp paired reads were retrieved for each library. Two biological replicates of mouse naive and CSR-activated B cells were performed for each genotype. Two repeats were performed in parental CH12F3 cell line along with two Ercc6l2−/− clones. One repeat was performed in parental U2OS cell line along with its isogenic Ercc6l2−/− cell line.
4
RNA-seq Library Preparation Protocols
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For infant control and CDM samples, RNA-seq libraries were prepared using the SMART-Seq version 4 ultralow input RNA kit (Clontech) per the manufacturer's instructions and sequenced using an Illumina HiSeq 2000. For PolyA-seq libraries, total RNA (130 ng) was used as a starting template, and libraries were generated as described (Batra et al. 2014 (link)) with the modification of adding barcodes to the library amplification primers to accommodate multiplex sequencing. For mouse P0 muscle and primary myoblast samples, RNA was isolated using the Direct-Zol RNA miniprep kit (Zymo Research). RNA-seq libraries were prepared from total RNA (500–700 ng) using the stranded RNA-seq kit with RiboErase (Kapa Biosystems) per the manufacturer's protocol. The CDM PolyA-seq and all mouse libraries were sequenced using an Illumina NextSeq 500.
5
RNA-seq of mESC Differentiation
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RNA from mESCs were harvested at several time points (Day 0 undifferentiated, 5 h after induction, 24 h APS/MPS, and 48 h DE/LM). RNA isolation and purification were performed using the Quick–RNA™ MicroPrep kit (Zymo Research), according to the manufacturer’s protocol. Integrity of purified RNA was checked on an Agilent Bioanalyzer using the RNA 6000 Pico Kit. Intact RNA was depleted of rRNA and prepared for sequencing using the KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems). Libraries were sequenced on the NextSeq 500 (Illumina), generating an average of 12–15 million reads per sample.
6
Sequencing-Based Transcriptome Analysis Protocol
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For each condition, triplicate total RNA was extracted with RNeasy kits (Qiagen), rRNA removed with Riboerase (Kapa Biosystems) and paired-end libraries were prepared with random hexamers (Kapa). Each library was sequenced to attain approximately 40M paired end 150bp reads on a NextSeq 500. Sequences were mapped to hg19 using STAR (Dobin et al., 2012 (link)), Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were determined with cufflinks (Trapnell et al., 2010 (link)), and differential gene expression was determined using featureCounts (Liao et al., 2014 (link)) and DESeq2 (Love et al., 2014 (link)).
7
RNA-Seq Library Preparation and Sequencing
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RNA libraries were generated, sequenced, and processed as described in [45 (link)]. RNA extraction was performed with the RecoverAll™ Total Nucleic Acid Isolation Kit (Invitrogen), following the manufacturer's protocol. RNA Integrity Number (RIN) was measured using Agilent 2100 bioanalyzer. RNA concentration was measured with Agilent RNA 6000 Nano or Qubit RNA Assay Kits. Depletion of ribosomal RNA was performed using RNA Hyper with RiboErase (KAPA Biosystem) Kit. Library concentrations and quality were measured with Qubit ds DNA HS Assay kit (Life Technologies) and Agilent Tapestation (Agilent). The samples were sequenced using Illumina HiSeq 3000 equipment for single end sequencing, 50 bp read length, for approximately 30 million raw reads per sample. Data quality check was conducted using Illumina SAV. De-multiplexing was performed using Illumina Bcl2fastq2 v 2.17 software [45 (link)].
8
Stranded RNA-seq Library Preparation
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RNA was isolated with Qiagen RNeasy columns according to the manufacturer’s protocol and 1000 ng of RNA were used to prepare a sequencing library for each sample using a Stranded RNA-seq kit with Ribo-Erase (KAPA Biosystems) (supplemental Table 14 ) (Key resources). Libraries were sequenced on a HiSeq4000, generating 100-bp reads.
9
Ribosomal RNA Depletion for Transcriptome Analysis
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Ribosomal RNA-depleted cDNA libraries were prepared using KAPA RNA HyperPrep kits with RiboErase (KAPA Biosystems) according to the manufacturer’s protocol. Libraries were sequenced with an Illumina Hi-seq 3000 (single-end, 1 × 100 bp).
10
Ribosomal RNA Depletion for Transcriptome Analysis
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