RNA quality was evaluated using Agilent Bioanalyzer 2100 Eukaryote Total RNA Pico chips. RNA-seq libraries were prepared using the SMART-Seq version 4 Ultra Low Input RNA Kit (Takara, 634889) from total RNA, following the manufacturer’s protocol. Libraries were then sequenced on a HiSeq 4000 using 75-bp paired end reads to an average depth of 22,445,650 ± 240,398 reads (SEM). Transcript abundance estimates were quantified using Salmon to mouse reference transcriptome from assembly GRCm38 (mm10), aggregated to gene level for UCSC-annotated genes using tximport, and DESeq2 was used to calculate normalized counts and differential expression [88 (link), 89 (link)]. CD4 and CD8 up/down gene signatures were generated through differential expression analysis via DESeq2. Differentially expressed genes (DEGs) between Yap-cKO versus WT CD4+ and CD8+ cells were defined as log2(FC) > 1 (up) or log2(FC) < −1 (down) and FDR < 0.05. DEGs were visualized with a heatmap combined with a barplot annotation, with the heatmap cells representing the log-normalized expression values for each sample. Each row is accompanied by a bar representing the log-fold change in gene expression (KO/WT). Plots were generated using the ComplexHeatmap software package available in R. Data have been deposited in NCBI GEO (accession number GSE139883).
Smart seq version 4 ultralow input rna kit
Manufactured by Takara Bio
The SMART-Seq version 4 ultralow input RNA kit is a laboratory tool designed for generating high-quality cDNA from small amounts of RNA input, including single cells. It utilizes a proprietary SMART (Switching Mechanism at the 5' end of the RNA Template) technology to amplify and convert RNA into full-length cDNA.
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Lab products found in correlation
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Tapestation high sensitivity d5000 screentape, by Agilent Technologies (1 mentions)
Hiseq 4000 sequencer, by Illumina (1 mentions)
Nextera xt index kit, by Illumina (1 mentions)
Agencourt ampure xp kit, by Beckman Coulter (1 mentions)
Nebnext multiplex oligos, by New England Biolabs (1 mentions)
Nebnext ultra 2 fs dna kit, by New England Biolabs (1 mentions)
Dnase treatment, by Qiagen (1 mentions)
Novaseq platform, by Illumina (1 mentions)
Riboerase, by Roche (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
High sensitivity dna bioanalyzer, by Agilent Technologies (1 mentions)
Qubit dsdna hs high sensitivity assay kit, by Agilent Technologies (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
M220 focused ultrasonicator, by Covaris (1 mentions)
Custom codeset, by NanoString (1 mentions)
Nsolver software, by NanoString (1 mentions)
7 protocols using smart seq version 4 ultralow input rna kit
1
Comprehensive RNA-seq Protocol for Differential Gene Expression Analysis
2
Single-cell RNA-sequencing Library Preparation
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cDNA was made from RNA and amplified using the SMART-Seq version 4 Ultra Low Input RNA Kit according to the manufacturer’s protocol (Takara Bio). Undiluted RNA samples from 2,000–8,000 cells were used for cDNA amplification with 14 cycles (Supplemental Table 2 ). The cDNA was purified by the Agencourt AMPure XP Kit (Beckman Coulter) and the quality assessed by TapeStation (High Sensitivity D5000 ScreenTape kit) according to the manufacturer’s protocols (Agilent Technologies). The samples were stored at –20°C before use.
Library preparation was performed using the Nextera XT DNA Library Preparation kit and the Nextera XT Index kit according to the manufacturer’s protocol (Illumina). The input cDNA was diluted according to the results from the TapeStation (Supplemental Table 2 ), and unique combinations of i5 (5 μL) and i7 (5 μL) index adapters were added to each respective sample. The libraries were assessed by TapeStation High sensitivity D5000 ScreenTape (Agilent Technologies), consisting of fragments mostly of 250–1,500 bp, with a peak of 600–1,000. Sequencing of the products was performed at the Bergen Genomics core facility, using a HiSeq 4000 sequencer according to the Illumina TruSeq Stranded mRNA protocol.
Library preparation was performed using the Nextera XT DNA Library Preparation kit and the Nextera XT Index kit according to the manufacturer’s protocol (Illumina). The input cDNA was diluted according to the results from the TapeStation (
3
Single-Cell RNA-Seq from Sorted Cells
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The RNA of 1000 freshly sorted cells was extracted using the Arcturus PicoPure kit (Applied Biosystems), and DNase treatment was performed (Qiagen). Subsequently, cDNA libraries were prepared using the SMART-Seq version 4 Ultra Low Input RNA kit (Takara Bio) with 12 cycles of amplification. Further, NEBNext Ultra II FS DNA kit and NEBNext Multiplex Oligos (New England BioLabs) were used to generate unique dual-barcoded sequencing libraries from cDNA libraries. To this end, 5 ng of cDNA library was fragmented for 22.5 minutes, adapters were ligated, and libraries were amplified for 8 cycles. RNA sequencing (RNA-seq) libraries were sequenced at a depth of 60 million reads, with a 100 bp paired end on a NovaSeq platform (Illumina).
4
RNA-seq Library Preparation Protocols
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For infant control and CDM samples, RNA-seq libraries were prepared using the SMART-Seq version 4 ultralow input RNA kit (Clontech) per the manufacturer's instructions and sequenced using an Illumina HiSeq 2000. For PolyA-seq libraries, total RNA (130 ng) was used as a starting template, and libraries were generated as described (Batra et al. 2014 (link)) with the modification of adding barcodes to the library amplification primers to accommodate multiplex sequencing. For mouse P0 muscle and primary myoblast samples, RNA was isolated using the Direct-Zol RNA miniprep kit (Zymo Research). RNA-seq libraries were prepared from total RNA (500–700 ng) using the stranded RNA-seq kit with RiboErase (Kapa Biosystems) per the manufacturer's protocol. The CDM PolyA-seq and all mouse libraries were sequenced using an Illumina NextSeq 500.
5
Single-Cell RNA-Seq Library Preparation
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SMART-Seq version 4 Ultra Low Input RNA kit from Clontech (catalog number 634892) was used. The kit generates Illumina compatible RNA-Seq libraries. Sorted single cells were cultured with and without treatment in the presence of cytokines until first cell division (40–44 hours). The daughter cells were manually separated, washed with phosphate-buffered saline (PBS), and collected for RNA sequencing. The cDNA synthesis and amplification was done as recommended by Clontech. The amplified cDNA generated from single cells were used to make libraries using Illumina’s Nextera XT DNA Library Preparation kit (catalog number FC-131-1096) as per Illumina’s instructions. The generated libraries were quantified using an agilent bio-analyzer, pooled, and subjected to next-generation sequencing in a Hi-Seq 2500 for pair-end 75 bp sequencing condition.
6
Isolation and RNA-sequencing of Tregs
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Tregs were isolated from either Foxp3EGFPcre or Foxp3EGFPCreRosa26N1c/+ mice by cell sorting, and their RNA was isolated using a Qiagen RNeasy Mini Kit (QIAGEN). RNA was then converted into double-stranded DNA (dsDNA) using SMART-Seq, version 4, Ultra Low Input RNA kit (Clontech). dsDNA was then fragmented into 200–300 bp sizes using an M220 Focused ultrasonicator (Covaris) and utilized for the construction of libraries for Illumina sequencing using a KAPA Hyper Prep Kit (Kapa Biosystems). Libraries were then quantified using a Qubit dsDNA HS (High Sensitivity) Assay Kit on an Agilent High Sensitivity DNA Bioanalyzer. Detailed procedures are described in Supplemental Methods .
7
Transcriptomic Profiling of Psoriatic Skin
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Library construction was done using SMART-Seq, version 4, Ultra Low Input RNA Kit (ClonTech, Mountain View, CA) and sequenced by Illumina (San Diego, CA) HiSeq 2000, at Beijing Genomic Institute (Hong Kong). P-value corresponds to differential gene expression test; corrections for false positive (type I errors) and false negative (type II) errors were performed using the Benjamini-Hochberg procedure (Reiner et al., 2003) . Data are presented for genes where the fold change of 2 or greater after treatment with a divergence of 0.8 or greater. Healthy skin (n ¼ 3), untreated psoriasis skin (n ¼ 3), and resolved psoriasis skin after ustekinumab treatment (n ¼ 3) were sent for analysis. For complete RNA sequencing data see https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi, GSE103489. Gene ontology pathways analysis has been performed through the DAVID platform (https://david.ncifcrf.gov) (Benjamini, 1995; Huang da et al., 2009) . NanoString counting is a quantitative method and was performed according to manufacturer's protocol (NanoString Technologies, Custom CodeSet). NanoString RNA counts were generated in nSolver software (NanoString Technologies, Inc.). Five samples per group of patients were analyzed, six for biologic treatment (infliximab, anti-TNF-a n ¼ 4; ustekinumab n¼2). The complete panel of transcripts analyzed and housekeepings are presented in Supplementary Table S2.
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