Isolated total, mRNP, and polysome RNA was purified via a RNeasy Mini Column (Qiagen) purification. After purification, 2 μl ERCC Spike in Mix 1 (Life Technologies) was added to approximately 100 ng of RNA from each sample. From these mixes, sequencing libraries were constructed using the Stranded Total RNA LT with Ribo-Zero TM Gold Library Prep kit (Illumina) and paired-end 100 bp reads sequenced on an Illumina HiSeq 2500 to a minimum depth of 30 million reads per sample. Reads were aligned to a testis transcriptome via RSEM [31 (link)].
Ercc spike in mix 1
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
The ERCC spike-in mix 1 is a set of synthetic RNA sequences designed to serve as internal controls for RNA-seq experiments. The mix contains a known quantity of external RNA control transcripts that can be spiked into samples to help assess the performance of the RNA-seq workflow.
Automatically generated - may contain errors
Lab products found in correlation
Truseq stranded mrna library prep kit high throughput, by Illumina (3 mentions)
Kapa library quantification kit, by Roche (3 mentions)
Bioanalyzer, by Agilent Technologies (3 mentions)
Qubit dsdna hs assay, by Thermo Fisher Scientific (3 mentions)
Hiseq 2500, by Illumina (2 mentions)
Truseq rna seq kit, by Illumina (2 mentions)
Novaseq sp full flow cell, by Illumina (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ribozero gold library prep kit, by Illumina (1 mentions)
Rneasy mini column, by Qiagen (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Dna free, by Thermo Fisher Scientific (1 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Rnaeasy extraction kit, by Qiagen (1 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions)
Masterpure yeast rna isolation kit, by Illumina (1 mentions)
19 protocols using ercc spike in mix 1
1
Comprehensive Transcriptome Analysis of Testis
2
Single-Cell RNA Lysis and RT-Primer Priming
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1000 HEK293 cells in 4.5 μl C1 wash buffer (Fluidigm) were lysed with 9 μl of lysis buffer (0.2% w/v Tween 20, 1 U/μl Promega RNAsin RNAse inhibitor, 2 μM reverse transcription primer, 2.5 mM dNTPs, 1× C1 loading reagent (Fluidigm), ERCC Spike-In Mix 1 at 20 000 molecules/cell (Life Technologies). The sample was incubated for 10 min at room temperature followed by 3 min at 70°C and 3 min at 10°C.
3
Single-Cell RNA Sequencing of Tumor and Normal Cells
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5 mL of STRT buffer (20 mM Tris-HCl at pH 8.0, 75 mM KCl, 6 mM MgCl2, 0.02% Tween-20) with 400 nM STRT-V3-T30 and 1:50,000 Life Technologies ERCC Spike-In Mix 1 were added into each well of the 96-well plate. The frozen tissues were thawed and digested into single cell suspension using 0.1% collagenase I (1mg/ml, 200U/ml) and 0.05% collagenase IV (0.1mg/ml, 20U/ml) for 2 hours under 37°C. Both single tumor cells and normal cells were sorted into the 96-well plate by Fluorescence-activated cell sorting (FACS) (75 tumor cells and 18 normal cells, 3 negative control). All of the following STRT steps including cDNA amplification and library construction were according to the protocols set by Saiful Islam et al. [17 (link)] The final cDNA library was sequenced on an Illumina HiSeq2000 using the customized sequencing primer STRT-SEQ-V3. Single-end reads of 50bp were generated along with 8-bp index reads corresponding to the cell-specific barcodes. The sequencing data from this study have been submitted to the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra ) under accession no. SRP078083.
4
Absolute Quantification of Transcripts
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Cells were lysed in TRI reagent (Sigma). RNA was then extracted with chloroform, precipitated with isopropanol and DNAse treated (DNA-free, Ambion). Total RNA (2 μg) was used per reverse transcription reaction. RNA was denatured in the presence of 2 μg of random hexamers (Invitrogen Life Technologies Ltd, Paisley, UK) for 5 minutes at 75°C, and reverse transcribed in a final volume of 40 μl with 200 U of SuperScript II (Invitrogen) at 42°C overnight followed by heat inactivation at 70°C for 15 minutes. Synthesized complementary DNAs were diluted in 300 μl of water and stored at −20°C until used.
Absolute quantification experiments were performed essentially as described in [39 (link)]. Briefly, 5.105 cells were lysed in 1 ml TRI reagent (Sigma) and 1 μl of a 1:20 dilution of ERCC Spike-in Mix#1 (Life Technologies Ltd, Paisley, UK) was added to each sample. After RNA extraction and reverse transcription, cDNAs were analysed by RT-qPCR. Transcript abundance were normalized to ERCC-00074 spike-in standard. ERCC-00096 and ERCC-00130 were also used as secondary controls.
Absolute quantification experiments were performed essentially as described in [39 (link)]. Briefly, 5.105 cells were lysed in 1 ml TRI reagent (Sigma) and 1 μl of a 1:20 dilution of ERCC Spike-in Mix#1 (Life Technologies Ltd, Paisley, UK) was added to each sample. After RNA extraction and reverse transcription, cDNAs were analysed by RT-qPCR. Transcript abundance were normalized to ERCC-00074 spike-in standard. ERCC-00096 and ERCC-00130 were also used as secondary controls.
5
Transcriptome Profiling of kdm2aa Mutants
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Using Sera Mag beads, total nucleic acid was isolated from 96 larvae from heterozygous sibling intercrosses for both kdm2aa alleles at 5 d.p.f. and 12 d.p.f. resulting in four experiments. KASP genotyping was performed on all samples to identify 4 individual homozygous mutant, heterozygous and wild-type sibling samples for each of the four experiments. From these 48 samples 300 ng total RNA were used to prepare sequencing libraries with Ambion ERCC spike-in mix 1 (Cat. No. 4456740) according to the manufacturer’s instructions using the Illumina TruSeq Stranded mRNA Sample Prep Kit Set A and B (RS-122-2101 and RS-122-2102). Paired end sequencing with a read length of 75 bp was performed on four lanes of Illumina HiSeq 2500 machines.
6
RNA-Seq Library Preparation with rRNA Depletion
7
RNA-Seq Library Prep with Illumina TruSeq
8
Human Myeloid Cell RNA-Sequencing
9
RNA-Seq Library Preparation with rRNA Depletion
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We prepared a library using 1 μg K-562 total RNA plus 2 μl of 1:100 diluted ERCC spike-in mix 1 (Ambion) using the TruSeq RNA-Seq kit (Illumina) with the following modifications. (1 ) The rRNA was depleted using the RNase H method19 (link) instead of using oligo (dT) selection. (2 ) We eluted the rRNA depleted RNA from the SPRI beads using EPF buffer from the TruSeq kit and heated at 70°C for 10 minutes. (3 ) We used a different set of barcoding indices rather than those in the TruSeq kit for the ligation and final PCR steps.
10
RNA-Seq Library Prep with Illumina TruSeq
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RNA sequencing library preparation was performed with the High Throughput TruSeq Stranded mRNA Library Prep Kit (Illumina) following the manufacturer’s protocol at half reaction volume. Input for each sample consisted of 300–500 ng of RNA and 5 μl of 1:500 diluted ERCC spike-in mix 1 (Ambion). Libraries were amplified for 12 cycles during the final amplification step. Libraries were quantified using the Qubit dsDNA HS assay (Thermo Fisher Scientific). Library size and quality were spot checked for a subset of samples by Bioanalyzer (Agilent). The average size of cDNA fragments in the libraries was 370 base pairs. Libraries were pooled at equimolar concentrations then the pool was quantitated using the KAPA library quantification kit (KAPA Biosystems). Libraries were sequenced single end 114 base pairs using NovaSeq_SP full flow cell (Illumina) at the Bauer Core Facility (Harvard University).
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