cDNA synthesis and amplification by LD-PCR were done following the User Manual of SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech, 634890), with some modifications under an open and clean workstation. Briefly for cDNA synthesis, the control total RNA (1 μg/μl in original concentration) was diluted to 4 × 10−6 μg/μl, and 3 μl of this was used to roughly match the RNA amount of a single cell (0.1 pg). In addition, 1 μl of external RNA controls consortium (ERCC) RNA Spike-In Mix (Invitrogen, cat. no. 4456740) as spike-in control was also added to all the samples and controls, for the final concentration of approximately 10,386 molecules/μl. This was followed by LD-PCR, as per manufacturer’s instructions.
Rna spike in mix
Manufactured by Thermo Fisher Scientific
The RNA spike-in Mix is a set of synthetic RNA molecules designed to be added to samples as internal controls for RNA-based experiments. The mix contains a defined concentration of various RNA transcripts that can be detected alongside the target RNA molecules in the sample. This allows for monitoring of RNA extraction, purification, and detection processes.
Automatically generated - may contain errors
Lab products found in correlation
Live dead cell viability assay, by Thermo Fisher Scientific (4 mentions)
Tapestation, by Thermo Fisher Scientific (2 mentions)
Microfluidic rna seq chip, by Standard BioTools (2 mentions)
Deltagene assays, by Standard BioTools (2 mentions)
Smart seq v4 ultra low input rna kit for sequencing, by Takara Bio (1 mentions)
Albacore 0, by Oxford Nanopore (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Smart seq stranded kit, by Takara Bio (1 mentions)
Quantseq 3 mrna seq library prep kit fwd, by Lexogen (1 mentions)
Rnase inhibitor, by Takara Bio (1 mentions)
Ultrapure water, by Thermo Fisher Scientific (1 mentions)
Affymetrix genechip primeview human gene expression array, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Dna free dnase 1, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Nucleospin gel purification kit, by Macherey-Nagel (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
14 protocols using rna spike in mix
1
Single-cell cDNA Synthesis and Amplification
2
Nanopore Sequencing of Soybean Transcriptome
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Sequencing data were basecalled using Albacore 0.8.4 (Oxford Nanopore Technologies). Reads were demultiplexed by barcode using porechop 0.2.0 (https://github.com/rrwick/Porechop , released 3/27/2017) with default settings. Reads were aligned for each sample to Glycine max_275_Wm82.a2.v1 primary transcripts (Schmutz et al., 2010 (link), available from phytozome.jgi.doe.gov) using the Burrows–Wheeler Aligner 0.7.15 (http://bio-bwa.sourceforge.net/ , released 6/1/2016) with the ‘ONT-2D’ option for long reads (Li and Durbin, 2009 (link)). BBMap pileup (37.28, https://sourceforge.net/projects/bbmap/ , released 6/1/2017) was used to compare aligned and reference transcript lengths, identify transcripts with at least one sequence alignment in all samples, and calculate GC content (%). The number of sequence reads at each base pair was calculated using SAMtools depth (1.4, http://www.htslib.org/ , released 13 March 2017). Due to limitations of Albacore 0.8.4, read depth varies across a transcript, especially in homopolymer stretches >5 bp, even when the complete molecule has been deeply sequenced. Sequence data from the External RNA Controls Consortium (ERCC) RNA Spike-In Mix (Invitrogen) showed that read depth varied by transcript abundance and that all 10 libraries were equally sequenced (see Supplementary Fig. S1 ).
3
Ribosome Profiling of S. cerevisiae
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Using S. cerevisiae strain yJC2229, ribosome footprint RNA and control total RNA were isolated and libraries were prepared as was previously described (Smith et al. 2014 (link)), with slight modifications. Specifically, cycloheximide treatment was omitted prior to cell harvest but was included during cell lysis. RNA purified from monosome fractions and control total RNA were depleted of ribosomal RNA once using the Yeast Ribo-Zero Gold rRNA Removal Kit (Illumina MRZY1324) according to the manufacturer's instructions following the addition of an RNA Spike-In mix (Thermo Fisher Scientific 4456740) to the total RNA sample. cDNA libraries were amplified using the indexed primer oKB690 (Smith et al. 2014 (link)) and were sequenced at the Case Western Reserve University Genomics Core Facility using the Illumina HiSeq2500 platform.
4
3' mRNA-Seq Library Preparation for Illumina
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Illumina compatible libraries were prepared from RNA using QuantSeq 3′ mRNA-seq Library Prep Kit FWD for Illumina (Lexogen GmbH, Wien, Austria) according to the manufacturer’s instructions. In brief, library generation was initiated by oligo-dT priming and first-strand synthesis. After RNA removal, libraries were subjected to random-primed second-strand synthesis. Illumina specific linker sequences are added by the primer, and the resulting double-stranded cDNA purified with magnetic beads. An additional 12 cycles of PCR amplification were carried out in order to introduce barcodes and to generate sufficient amounts of DNA required for cluster generation. After final purification, libraries were measured on TapeStation and Qubit (ThermoFisher, Waltham, MA) to determine quantity and size. The resulting libraries were on average 400-bp size with an average insert size of 270 bp. The method does not require prior poly(A) enrichment or ribosomal RNA depletion. ERCC (External RNA Controls Consortium) RNA Spike-In Mix (Cat# 4456740 Thermo Fisher Scientific, Waltham, MA) was added to the RNA before library preparation to allow inter-sample normalization and control for variabilities.
5
RNA-seq Library Construction and Sequencing
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The total RNA-seq libraries were constructed using the SMART-Seq Stranded Kit (Takara) as previously described.18 (link) For each library, 1μL 1/10000 ERCC RNA spike-in Mix was included prior to RNA shearing (Thermo Fisher Scientific). Sequencing (2 × 150bp) was performed on the Illumina NovaSeq 6000 platform at Novogene. Library information is available in Table S4 .
6
Cerebellar RNA Sequencing Protocol
7
Single-cell RNA-seq of human fetal brain
8
Single-Cell RNA-Seq of Tau-EGFP Neurons
9
RNA Sample Preparation and Gene Expression Analysis
10
Quantifying HIV-1 Transcript Abundance
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After adding ERCC (External RNA Controls Consortium) RNA Spike-In Mix (Ambion, Life Technologies), RNA was fragmented and cDNA prepared as previously described (Eckwahl et al. 2015 (link)). Libraries were amplified with 20 PCR cycles and purified using the Nucleospin gel purification kit (Machery-Nagel). Samples were quantitated using the KAPA library quantification kit (Kapa Biosystems), spiked with 20% PhiX Sequencing Control (Invitrogen), and sequenced for 75 bases on an Illumina HiSeq 2500 at the Yale Center for Genome Analysis. After demultiplexing libraries based on the index sequences, adapters were removed (FASTX-toolkit) and reads aligned using the Burroughs-Wheeler Alignment (BWA) tool (Li and Durbin 2009 (link)). After filtering reads that mapped to PhiX and ERCC controls, the remaining reads were mapped to the HIV-1 genome (AF324493.2) and then to the human hg19 assembly. BEDTools (Quinlan and Hall 2010 (link)) was used to assign reads to genomic features, while Sailfish (Patro et al. 2014 (link)) was used to determine transcripts per million (TPM) (Li et al. 2010 (link)). To examine the extent to which full-length repeat transcripts could be present, reads were multimapped using Bowtie (Langmead et al. 2009 (link)), allowing ≤3 mismatches.
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