QuantSeq 3’ end counting was used for polysome profiling samples as well as matched wild-type, Ddx6 KO, and Dgcr8 KO mRNA samples (Figure 5C ). RNA was isolated using RNeasy Micro kits (Qiagen). RNA-Seq libraries were generated using the QuantSeq 3’ FWD kit (Lexogen) and sequenced with single-end 50 bp reads.
Quantseq 3 fwd kit
Manufactured by Lexogen
The QuantSeq 3' FWD kit is a library preparation kit designed for 3' mRNA sequencing. It enables the generation of sequencing-ready libraries from total RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy micro kit, by Qiagen (1 mentions)
Allprep protocol, by Qiagen (1 mentions)
Novaseq 6000, by Lexogen (1 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Gradient station, by BioComp Instruments (1 mentions)
Zymo clean and concentrator 5 kit, by Zymo Research (1 mentions)
3 protocols using quantseq 3 fwd kit
1
Polysome Profiling with QuantSeq
2
RNA-seq Analysis of Tumor Samples
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Total RNA was extracted for every tumor sample using the QIAGEN AllPrep protocol.14 Overall, 150 ng of total RNA was used to generate RNAseq sequencing library using the Lexogen QuantSeq 3′ FWD kit and sequenced by the Institut du cerveau iGenseq plateform on a NovaSeq 6000 aiming for a minimum of 10 million reads. Raw RNAseq reads were mapped to the human genome (Ensembl GRCH38) and Ensembl's reference transcriptome using STAR.15 (link) Gene counts were obtained using FeatureCount,16 (link) normalized by a Upper Quartile procedure, and logged on a base 2. The improved GemPred signature13 (link) was applied to the normalized counts to dichotomize in a GemPred+ or GemPred– status for each sample.
3
Polysome Profiling of Mouse ESCs
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Two plates of 6 million V6.5 ESCs were seeded in a 15 cm plate 48 hr prior to collection (Eggan et al., 2001 (link)). Cells were incubated with 100 ug/ml cycloheximide (Sigma) for 2 min and then moved to ice. Cells were washed and scraped in PBS with cycloheximide, spun down, and then lysed. Lysate was loaded onto a 10–50% sucrose gradient and centrifuged at 35,000 RPM for 3 hr. Gradients were collected on a gradient station (Biocomp). For each sample, the monosome, low polysome (2–4 ribosomes), and high polysome (4 + ribosomes were collected). RNA was extracted from gradient fractions with TRIzol LS (Invitrogen) and concentrated with the Zymo Clean and Concentrator-5 kit (Zymo) prior to library preparation with the QuantSeq 3’ FWD kit (Lexogen).
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