RNA-seq libraries were prepared from 50 ng of total RNA using the NEBNext Single Cell/Low Input RNA library Prep Kit for Illumina and sequenced on an Illumina NovaSeq6000 S-prime flow cell with paired-end 50-bp reads, yielding 40–90 million clusters per library. For DART-seq, 50 ng of RNA was used for in vitro deamination as previously described (35 (link)) with some modifications. RNA was incubated with 250 ng of recombinant APOBEC1-YTH-HA protein for 4 h at 37 °C in 1x deaminase buffer (10 mM Tris–HCl, pH7.5, 50 mM KCl, 0.1 μM ZnCl2). RNA was purified with QIAGEN RNeasy MinElute Cleanup kit and used as input for RNA-seq libraries as above. RIP-seq libraries were prepared from 9 ng of RNA using the SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input Mammalian (Takara) for three biological replicates. For each replicate, the lysates of three wells of neurons grown in a six-well plate were pooled and then equally split for YTHDF1, YTHDF2 and YTHDF3 RIPs. This enabled 3 YTHDF RIPs (one each for YTHDF1,2, and 3) from a single shared input sample. Libraries were sequenced on a single lane of a NovaSeq6000 S-prime with paired-end 50-bp reads, yielding 18–54 million clusters per library.
Smarter stranded total rna seq kit v3 pico input mammalian
Manufactured by Takara Bio
Sourced in Japan
The SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian is a lab equipment product designed for RNA-sequencing applications. It is capable of generating strand-specific libraries from small amounts of total RNA derived from mammalian samples.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (5 mentions)
Nextseq 550, by Illumina (3 mentions)
Nextseq 2000, by Illumina (2 mentions)
Qubit dsdna hs, by Agilent Technologies (2 mentions)
Agilent 2100 bioanalyzer high sensitivity dna, by Agilent Technologies (2 mentions)
Ho 150 kit, by Illumina (2 mentions)
Facsaria fusion, by BD (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000 s prime flowcell, by Illumina (1 mentions)
Nebnext single cell low input rna library prep kit, by Illumina (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy ffpe kit, by Qiagen (1 mentions)
Nextseq 5000, by Illumina (1 mentions)
Quick rna microprep kit, by Zymo Research (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Agilent high sensitivity rna screentape, by Agilent Technologies (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Dna high sensitivity kit reagents, by Agilent Technologies (1 mentions)
18 protocols using smarter stranded total rna seq kit v3 pico input mammalian
1
Transcriptome Analysis: RNA-seq, DART-seq, and RIP-seq
2
RNA-seq Analysis of Mammalian Transcriptome
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RNA extractions were performed using the Direct-zol RNA Microprep kit (R2061) according to the manufacturer’s instructions. RNA library preparation was realized following the manufacturer’s recommendations (SMARTer Stranded Total RNA-Seq Kit v3—Pico Input Mammalian from Takara). Final samples pooled library preparations were sequenced on Nextseq 2,000 ILLUMINA with a P2-200 cycles cartridge (2 × 400 millions of 100 base reads) corresponding to 2 × 66 millions of reads per sample after demultiplexing. Differential analysis was done with the DESeq2 method and gene ontology (GO) enrichment using the Gene Set Enrichment Analysis (GSEA) in the Quby-RNA tool of the Data Analysis Core facility at the Paris Brain Institute.
Volcano plots, heatmaps, and bubble plots were generated using the open-source web apps VolcaNoseR (Goedhart and Luijsterburg, 2020 (link)), Heatmapper (Babicki et al., 2016 (link)), and SRPlot (https://www.bioinformatics.com.cn/en ), respectively.
Volcano plots, heatmaps, and bubble plots were generated using the open-source web apps VolcaNoseR (Goedhart and Luijsterburg, 2020 (link)), Heatmapper (Babicki et al., 2016 (link)), and SRPlot (
3
RNA-seq Analysis of SNRPB Knockdown
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RNA from control and SNRPB-KD MeWo and A375 samples was collected and isolated as described above. RNA-seq libraries were prepared using SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara) according to manufacturer’s recommendations, using 10 ng total RNA as input. The libraries were sequenced on a 2x 35 bp PE run on a NextSeq 550 instrument (Illumina). UMI-tools (v1.0.0) was used to extract UMI’s from read 2. Cutadapt (v2.3) was used to trim R1 to an identical length. This is required since MISO expects reads of equal length for both pairs. STAR (v2.7.1a) was then used to align reads to the human genome (hg38) and UMI collapse (1.0.0) was used to remove PCR duplicates (with --two-pass --paired --remove-unpaired --remove-chimeric flags). The resulting deduplicated bam files were sorted by name and converted back to fastq files (samtools v1.7). The deduplicated fastq files were aligned to a curated human transcriptome using bowtie (2.3.5) and bamPEFragmentSize (3.5.1) was used to calculate fragment length distribution parameters (i.e. mean and standard deviation). MISO (0.5.4) was then used to analyze changes in skipped exon inclusions by passing the deduplicated STAR-aligned bam files along with the fragment size distribution for each sample.
4
FFPE RNA Extraction and Sequencing Protocol
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Total RNA was extracted from newly cut 9–10 μm FFPE adjacent to unstained sections using the RNeasy FFPE kit (Qiagen, France) according to the manufacturer’s protocol using 4–6 sections per case depending on the different ROIs : Ac/ILF-, Ac/ILF+ and ELF.
RNA yield and quality were determined by UV absorption on a NanoDrop One spectrophotometer (ThermoScientific®). Approximately 25µL of total RNA (1.5 ng/µL) from each sample was used for RNA Sequencing.
RNA library preparation followed manufacturer’s instructions (SMARTer Stranded Total RNA-Seq Kit v3—Pico Input Mammalian from Takara).
Final samples pooled library prep were sequenced on ILLUMINA Novaseq 6000 with S1-200 cartridge (2 × 1600Millions of 100 bases reads), corresponding to 2 × 38Millions of reads per sample after demultiplexing.
This work used equipment and services from the iGenSeq core facility (Genotyping and sequencing), at ICM (Institut du Cerveau et de la Moelle épinière, Paris).
RNA yield and quality were determined by UV absorption on a NanoDrop One spectrophotometer (ThermoScientific®). Approximately 25µL of total RNA (1.5 ng/µL) from each sample was used for RNA Sequencing.
RNA library preparation followed manufacturer’s instructions (SMARTer Stranded Total RNA-Seq Kit v3—Pico Input Mammalian from Takara).
Final samples pooled library prep were sequenced on ILLUMINA Novaseq 6000 with S1-200 cartridge (2 × 1600Millions of 100 bases reads), corresponding to 2 × 38Millions of reads per sample after demultiplexing.
This work used equipment and services from the iGenSeq core facility (Genotyping and sequencing), at ICM (Institut du Cerveau et de la Moelle épinière, Paris).
5
Mammalian Total RNA-seq Using SMARTer
6
SF3A1 Overexpression RNA-seq
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RNA-seq was done on SF3A1 over-expression and control cell lines. RNA was extracted from samples by column clean up (Zymo Quick-RNA Microprep Kit). RNA-seq libraries were prepared from these samples using the SMARTer® Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara) kit according to manufacturer’s instructions. Sequencing was performed on an Illumina NextSeq 5000.
7
Mammalian Total RNA-seq Using SMARTer
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Full-length total RNA-seq was conducted using the SMARTer Stranded Total RNA-seq Kit v3, Pico Input Mammalian (Takara Bio, catalog no. 634485). We used 1,000 pg of total RNA for Input samples. For RiboTag IP samples, an estimated 500–1,000 pg (via ActB qPCR) (see Figure S1I ) was used. Libraries were constructed according to the manufacturer’s instructions with the following parameters: (1) 4-min fragmentation before reverse transcription, and (2) 14–15 cycles of PCR after ZapR depletion. Unique dual indexes were assigned to each sample, and libraries were pooled at 1 nM after quantification using Qubit dsDNA HS and Agilent 2100 Bioanalyzer High Sensitivity DNA assays. Pooled libraries were sequenced on a NextSeq 500 with 2 × 75 bp paired end reads (HO 150 kit, Illumina).
The first 15 bp of Read 2 (UMI and TSO sequences) were removed using fastx-trimmer (http://hannonlab.cshl.edu/fastx_toolkit/index.html ), and paired-end reads then were depleted of rRNA by alignment to mouse 5S, 5.8S, 18S, and 28S rRNA using bowtie2 (Langmead and Salzberg, 2012 (link)). rRNA-depleted paired-end reads were then aligned to the mouse genome (GENCODE M25, GRCm38.p6) using STAR 2.6.7a (Dobin et al., 2013 (link)). Uniquely mapped reads were then quantified at the exon level using feature-Counts version 1.6 (Liao et al., 2014 (link)).
The first 15 bp of Read 2 (UMI and TSO sequences) were removed using fastx-trimmer (
8
RNA Extraction and Sequencing Protocol
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Sorted cells or cell pellets were lysed in 350μL of RLT buffer (QIAGEN), and total RNA was extracted with RNeasy micro kit (QIAGEN, Cat# 74004). Libraries were constructed with SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara Cat# 634486) following the manufacturer’s instructions. For bulk RNAseq sample details please refer to Table S3 .
9
Profiling Mtb-infected Macrophages
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Single cell suspensions from lung were analyzed and sorted by fluorescence-activated flow cytometry (FACS) into Mtb-infected (mCherry+) and bystander (mCherry-) MDM (dead- SiglecF- Ly6G- CD11b+ CD64+) populations. RNA was isolated using Trizol, and quantified using bulk RNA-seq (Psomagen) after construction of Illumina sequencing libraries using the SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara). Noise from low-expression transcripts was filtered, and analysis of differentially expressed genes (DEGs) across groups was done using the edgeR module in R(27 (link)).
10
Sequencing of Mammalian Total RNA
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Sequencing libraries were constructed with SMARTer® Stranded Total RNA-Seq Kit v3–Pico Input Mammalian (Takara Bio) according to the manufacturer’s instructions. Three nanograms of total RNA were used as input. Library size was determined by Agilent High Sensitivity RNA ScreenTape (Agilent Technologies). Library concentration was determined by the KAPA Library Quantification Kit (Kapa Biosystems) according to the manufacturer’s instructions. Paired-end sequencing was performed with NextSeq 1000/2000 P2 Reagents (100 Cycles) v3 on a NextSeq 2000.
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