Preparation of total RNA-seq libraries was performed using SMART-seq Stranded Kit (Clontech), according to the manufacturer’s instructions. In brief, 30 ZP-free two-cell, four-cell and eight-cell stage embryos whose cleavage stages were visually confirmed under the microscope, were lysed in 1× Lysis Buffer containing RNase inhibitor (0.2 IU µl−1, from SMART-seq Stranded Kit, Clontech), directly. RNAs were randomly sheared by heating at 85°C for 8 min and subjected to reverse transcription with random hexamers and PCR amplification. Ribosomal fragments were depleted from each cDNA sample with scZapR and scR-Probes. Indexed total RNA-seq libraries were enriched through a second PCR amplification and sequenced using an Illumina HiSeqX sequencer (paired end, 150 bp). Two biological replicates were generated for each sample.
Smart seq stranded kit
Manufactured by Takara Bio
Sourced in Japan, United States
The SMART-Seq Stranded Kit is a technology for full-length, single-cell RNA sequencing that generates strand-specific data. It enables the preparation of high-quality libraries from nanogram-level inputs of total RNA or purified poly(A)+ RNA.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (11 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (6 mentions)
Nextseq 500, by Illumina (4 mentions)
Nextera xt dna library preparation kit, by Illumina (3 mentions)
4200 tapestation system, by Agilent Technologies (3 mentions)
Nextseq 550, by Illumina (3 mentions)
Rneasy micro kit, by Qiagen (3 mentions)
Agilent dna 1000 kit, by Agilent Technologies (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Kapa library quantification kit, by Roche (3 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (3 mentions)
Nextseq 550 platform, by Illumina (2 mentions)
Nextseq 500 550 platform, by Illumina (2 mentions)
Smart seq v4 ultra low input rna kit, by Takara Bio (2 mentions)
Direct zol rna microprep kit, by Zymo Research (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Tapestation 4200, by Agilent Technologies (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
53 protocols using smart seq stranded kit
1
Total RNA-seq Library Preparation from Staged Embryos
2
RNA-Seq Analysis of Kidney Organoids
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For RNA sequencing, total RNA was extracted as described above. The samples preserved at −80 °C were shipped and analyzed by DNAFORM. The quality of total RNA was evaluated by a Bioanalyzer (Agilent) to ensure over 8.0 RIN (RNA integrity number) or by electrophoresis waveforms. Double-stranded cDNA libraries (RNA-seq libraries) were prepared using a SMART Seq Stranded Kit (Clontech) according to the manufacturer’s protocols. RNA-seq libraries were sequenced using paired-end reads (50 nt of read 1 and 25 nt of read 2) on a NextSeq 500 (Illumina). Obtained reads were mapped to the human GRCh38 genome analyzed by STAR (version 2.7.3a). Annotated reads were counted using featureCounts (version 2.0.1) and RSEM (version 1.3.1). FPKM values were calculated from mapped reads by normalizing to total counts. The gene expression heatmap was drawn based on the log2 fold change (log2FC) compared with day 13 + 4 kidney organoid samples using FPKM.
3
Total RNA-seq and Poly(A) RNA-seq Library Preparation
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Total RNA-seq libraries were prepared using the SMART-Seq Stranded Kit (Clontech) following the manufacturer’s instructions. Poly(A) RNA-seq libraries were prepared as previously described (56 (link)) using a SMARTer ultralow input RNA cDNA preparation kit (Clontech). A Nextera XT DNA library preparation kit (Illumina) was used for poly(A) RNA-seq cDNA fragmentation, adaptor ligation, and amplification according to the manufacturer’s instructions. The prepared RNA-seq libraries were sequenced on NextSeq 550 (Illumina) with paired-ended 75-bp reads.
4
Total-RNA-Seq Library Preparation and Sequencing
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After RNA extraction, a RNA quality control was performed with the 4200 Tape Station system (Agilent). Only RNAs having an RNA Integrity Number (RIN) greater than 6 were used for library preparations. Total-RNA-Seq library preparation was performed starting from 0.5 ng of total-RNA with the SMART-Seq Stranded Kit (Clontech-Takara). Libraries obtained were qualitatively assessed by using TapeStation 4200 (Agilent) and quantified by Qubit Fluorimeter (Thermo Fisher Scientific). Afterward, they were multiplexed in equimolar pools and sequenced on a NextSeq-550 Illumina Platform generating at least 60 million 75bp-paired-end reads per sample.
RNA-Seq data are available at GEO under accession number GSE160362.
RNA-Seq data are available at GEO under accession number GSE160362.
5
Total RNA-seq Library Preparation
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Total RNA-seq library preparation was performed starting from 0.5 ng of total RNA with the SMART-Seq Stranded Kit (Clontech–Takara). Libraries were obtained and qualitatively assessed by using the Agilent 4200 TapeStation and quantified by Qubit Fluorimeter. Afterward, they were multiplexed in an equimolar pool and sequenced on an Illumina NextSeq 500 Platform, generating at least 40 million 75-bp–PE reads per sample (i.e., 80 million reads/sample).
6
Transcriptome Profiling of CD8+ T Cells
7
RNA-seq of FACS-sorted CD158b1b2j-neg NK cells
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FACS-sorted CD158b1b2jneg NK cells were lysed in 49 μL of RLT buffer (Qiagen) containing 1 μL of RNAse inhibitor (Thermo Fisher Scientific) and stored at -80°C.
The MicroRNAeasy KitTM with DNAse (Qiagen) was used to purify total RNA, which was then quantified by a Nanodrop 2000 (Thermo Fisher Scientific). Starting from 0.5 ng of high-quality total RNA with an RNA Integrity Number (RIN) greater than 6, assessed using a 4200 Tape Station (Agilent), libraries were prepared with the SMART-Seq Stranded Kit (Clontech-Takara). Libraries were then multiplexed in equimolar pools and sequenced by using a NextSeq-550 Illumina Platform. At least 60 million 75bp-paired-end reads per sample were generated. Read alignment, differential gene expression, and functional enrichment analyses were performed as previously described (16 (link)).
The MicroRNAeasy KitTM with DNAse (Qiagen) was used to purify total RNA, which was then quantified by a Nanodrop 2000 (Thermo Fisher Scientific). Starting from 0.5 ng of high-quality total RNA with an RNA Integrity Number (RIN) greater than 6, assessed using a 4200 Tape Station (Agilent), libraries were prepared with the SMART-Seq Stranded Kit (Clontech-Takara). Libraries were then multiplexed in equimolar pools and sequenced by using a NextSeq-550 Illumina Platform. At least 60 million 75bp-paired-end reads per sample were generated. Read alignment, differential gene expression, and functional enrichment analyses were performed as previously described (16 (link)).
8
Transcriptome Profiling of CD8+ T Cells
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RNA was extracted from 50,000 FACS-purified CD8+ T cells per subset using a Direct-Zol RNA Microprep Kit (Zymo Research) and stored at −80°C. Quality control was performed using a High Sensitivity RNA ScreenTape Assay with a 4200 TapeStation System (Agilent). Libraries for mRNA sequencing were prepared from 5 ng of total RNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara). Full-length cDNAs were processed using a Nextera XT DNA Library Preparation Kit (Illumina). Quality control was performed using a High Sensitivity DNA ScreenTape Assay with a 4200 TapeStation System (Agilent). Libraries were then multiplexed in an equimolar pool and sequenced using a NextSeq 500/550 Platform (Illumina). An average of 11 million single-end 75 base pair (bp) reads were generated per sample. Libraries for total RNA sequencing were prepared from 1 ng of total RNA using a SMART-Seq Stranded Kit (Clontech-Takara). Quality control and sequencing were performed as described for the mRNA libraries, generating an average of 103 million paired-end 75 bp reads per sample.
9
RNA Isolation and Sequencing Library Preparation
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RNA was isolated with the RNEasy Plus Mini Kit (QIAGEN) according to manufacturer’s recommendations. cDNA was generated and amplified with the SMARTer Ultra Low Input RNA kit for Illumina Sequencing (Clontech Laboratories, Inc.) or the SMART-Seq Stranded Kit (Takara Bio) according to the manufacturer’s protocol. For ultra-low applications, sequencing libraries were prepared using the NEXT ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs) according to the manufacturer’s instructions with the following modifications: The adaptor-ligated double-stranded cDNA (10μl) was amplified using NEBNext Multiplex Oligos for Illumina (New England Biolabs, 25 μM primers), NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs) and 15 cycles of PCR. For all samples, final libraries were validated using Agilent 2100 Bioanalyzer (Agilent Technologies) and Qubit fluorometer (Invitrogen), normalized and pooled in equimolar ratios. 50bp single-read sequencing was performed on the Illumina HiSeq™ 2000 v4 or 75bp single-read sequencing on the Illumina Nextseq™ 500 according to the manufacturer’s protocol.
10
RNA-seq Analysis of Mouse Transcriptome
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Total RNA was obtained from each FGO, and RNA-seq libraries were prepared using SMART-Seq Stranded Kit (Takara Bio) according to the standard protocol (Ramskold et al, 2012 (link)). In brief, total RNA was fragmented at 85°C for 6 min and then processed under the ultra-low-input workflow. PCR1 was performed for 10 cycles, and PCR2 was performed for 12 cycles. The final cleanup was performed twice. The libraries were sequenced on an Illumina NovaSeq 6000 using SP Reagent Kit (paired-end 151 nucleotides). Reads were trimmed and mapped to the reference mouse genome (mm10) by HISAT2 v2.1.0 (Kim et al, 2019 (link)). Transcripts were assembled by StringTie v2.1.3 (Kovaka et al, 2019 (link)). For hierarchical clustering and identification of the differentially expressed genes, iDEP online tools were used (Ge et al, 2018 (link)). Transcripts were filtered out by the criteria of at least 0.5 counts per million in all samples.
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