Total RNA was isolated from FACS-sorted CD11bhighLy6Ghigh neutrophils using an RNAeasy Plus Micro kit (Qiagen). RNA integrity was verified by a Bioanalyzer (Agilent). Sample cDNAs were prepared using the SMART-Seq v4 Ultra Low Input kit (Takara) using 250 pg of input total RNA followed by library preparation using a TruSeq DNA Nano kit (Illumina). Libraries were verified by Tapestation (Agilent). Library concentrations were determined by real-time PCR with a StepOnePlus Real Time PCR System (Thermo Fisher) and a Kapa Library Quantification Kit (Kapa Biosystems/Roche). Libraries were sequenced with a 100 cycle single read protocol on a HiSeq 2500 (Illumina) with four libraries per lane. Fastq files were assembled using Bcl2Fastq2 (Illumina).
Smart seq v4 ultra low input kit
Manufactured by Takara Bio
Sourced in United States, Japan
The SMART-Seq v4 Ultra Low Input kit is a library preparation kit designed for generating high-quality cDNA libraries from ultra-low input RNA samples. The kit utilizes the SMART (Switching Mechanism at 5' end of RNA Template) technology to generate full-length cDNA from as little as 10 pg of total RNA.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt dna library preparation kit, by Illumina (4 mentions)
Bioanalyzer, by Agilent Technologies (3 mentions)
Rneasy plus micro kit, by Qiagen (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Truseq nano dna kit, by Illumina (2 mentions)
Rnaeasy plus micro kit, by Qiagen (2 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Tapestation, by Agilent Technologies (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Nextera xt kit, by Illumina (2 mentions)
Rlt plus buffer, by Qiagen (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Direct zol rna kit, by Zymo Research (2 mentions)
Truseq rna library prep kit, by Illumina (2 mentions)
Thruplex dna seq kit, by Takara Bio (2 mentions)
Nextseq, by Illumina (2 mentions)
Ovation rna seq system v2, by Tecan (2 mentions)
Bcl2fastq2, by Illumina (1 mentions)
17 protocols using smart seq v4 ultra low input kit
1
Neutrophil Transcriptome Profiling
2
Drosophila per RNA-seq Data Analysis
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All steps required for the RNA integrity check, library preparation and sequencing were performed by experts at the iGE3 Genomics Platform at the University of Geneva. Total RNA integrity and quantitation was performed using the Bioanalyzer High Sensitivity RNA 6000 Pico kit (Agilent, Cat. # 5067-1513). cDNA library preparations were conducted using the SMART-Seq v4 Ultra Low Input kit from TaKaRa (Cat. # 634894) according to the manufacturer’s instructions. Libraries were sequenced on the HiSeq2500 Illumina machines using single-end 50-bp reads to generate more than 10 million reads per library. The iGE3 Genomics Platform performed QC, trimming of sequencing adaptors, and filtering of low-quality reads. Raw reads were aligned with the Dm3 Drosophila genome using the EPFL’s HTSstation platform pipeline92 (link). Because HTSstation platform has been discontinued, for per0 RNA-seq data analysis, raw reads were aligned with the Dm6 Drosophila genome using STAR software (v2.7.9a). The raw counts of reads associated with each of the exons were determined using featureCounts function of the Rsubread package (v1.22.2). All libraries generated on average ~18 million single-end 50 bp reads, from which approximately 80% (~14 million reads) were mapped to the genome. The normalized expression levels (Reads Per Kilobase of Million mapped reads; RPKM) were used for downstream analysis.
3
Microglia Isolation and RNA-seq Analysis
4
Neutrophil RNA-seq from Mouse Whole Blood
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Whole blood was isolated from mice by cardiopuncture, and total RNA was isolated from FACS-sorted CD11bhighLy6Ghigh neutrophils using an RNAeasy Plus Micro kit (Qiagen). Integrity of RNA was verified by an Agilent Bioanalyzer prior to cDNA preparation using the SMART-Seq v4 Ultra Low Input kit (Takara). RNA libraries were prepared using a TruSeq DNA Nano kit (Illumina) and verified by Tapestation (Agilent). Library concentrations were measured by real-time PCR with StepOnePlus Real Time PCR System (ThermoFisher) followed by a Kapa Library Quantification Kit (KapaBiosystems/Roche). Libraries were then sequenced with a 100-cycle single-read protocol using a HiSeq 2500 sequencer (Illumina). Quality control checks were performed using the FastQC package, and raw reads were normalized using the DESeq2 Bioconductor package.
5
Transcriptome Sequencing from Nanogram RNA
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Libraries for RNAseq were prepared from 3 ng of RNA, by first generating full length double stranded cDNA (ds cDNA) using the SMART-Seq v4 Ultra™ Low Input kit (Cat. 634891, Takara Bio USA) followed by Illumina’s Nextera XT DNA Library Preparation kit (FC-131–1096, Illumina Inc. San Diego, CA). Briefly, 3 ng of RNA were used to obtain first strand cDNA using SMART-Seq2 template switching and extension with SMARTScribe™ reverse transcriptase. cDNA was amplified using 10 cycles of PCR with SeqAmp DNA polymerase. The resulting dscDNA was validated by determining average size (~ 2 kb) on an Agilent Bioanalyzer high sensitivity chip. 500 pg of the dscDNA were tagmented with the Nextera transposome to fragments of ~ 350 bp containing adapter sequences. Unique Indexes for each library were added by using 12 cycles of PCR amplification and resulting libraries were pooled together for sequencing. The pools were clustered at 6.5 pM on single read flow cell and sequenced for 50 cycles on an Illumina HiSeq 2500. Base call files generated from the sequencer were demultiplexed and converted to FASTQ files using the Illumina CASAVA 2.17 pipeline. Quality of the raw sequence reads was assessed with FastQC (Version 0.10, Babraham Bioinformatics) and FASTX toolkit (version 0.0.13, http://hannonlab.cshl.edu/fastx_toolkit/ ).
6
Microglia Isolation and RNA-seq Analysis
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20,000 microglia were isolated from either hemibrains or micro-dissected hippocampi as described above, and sorted into RLT Plus buffer (Qiagen) containing beta-mercaptoethanol. RNA was extracted using a RNeasy Micro Plus kit (Qiagen) according the manufacturer’s protocol. RNA integrity was assessed on a Bioanalyzer (Agilent), and high quality samples were used for library preparation. cDNA synthesis and amplification was performed using the SmartSeq v4 Ultra-low input kit (Takara), and libraries were tagmented, adaptor ligated, and indexed using the Nextera XT kit (Illumina). After normalization and pooling, libraries were sequenced on a Hiseq 4000 (Illumina) using paired-end 100bp reads. Libraries were sequenced to a depth of >30 million reads per sample. Raw sequencing files were demultiplexed with bcl2fastq, reads were aligned using STAR, the count matrix was generated using SummarizedExperiment, and differential expression analysis was performed using DESeq2 with standard settings.
7
Single-cell RNA-seq Library Preparation
8
Quantifying Gene Expression in Sorted Nuclei
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RNA concentration from sorted nuclei was measured in Qubit 2.0 (Invitrogen) using RNA high-sensitivity kit. Three nanograms of RNA was used to prepare cDNA using SMART-Seq v4 Ultra Low Input Kit (Takara). Following cDNA preparation, equal amounts of cDNA were subjected to qPCR using primers (designed by Roche universal probe library tool) for Chst3 mRNA. A standard curve was generated to calculate primer efficiency, which was used to obtain the normalized expression value (delta–delta Ct method), using DNA topoisomerase I (Top1) as a housekeeping gene [44 (link)]. Relative expression was calculated by normalizing to the 2-3 M samples. A list of probes used for sorted nuclei qPCR is shown in Table 2 .
9
Bulk RNA-seq Protocol for Transcriptomics
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Bulk RNA-seq: For each group, 104-106 cells were lysed in Trizol lysis buffer. RNA was extracted and processed using a QIAGEN miRNEasy Extraction kit. RNA library construction was done using a SMART-Seq v.4 Ultra-low input kit (Takara Bio, Shiga, Japan) and cDNA was converted to sequencing library using NexteraXT DNA Library Prep Kit with indexing primers (Illumina, San Diego, CA). For transcriptomics, libraries were sequenced for single end 1x100bp reads at 30 million reads/sample on a NOVASeq 6000 (Illumina). Quality controlled reads (fastq) were aligned to mouse genome (mm10) using HISAT2 and BAM file conversion, sorting, and indexing were done with Samtools (24 (link), 25 (link)). Read counts were obtained from resulting BAM files using Stringtie (26 (link)). For transcriptomics analysis, differentially expressed genes were identified using DESeq2 (FDR<.05) (27 (link)). FPKM values from Stringtie of most differentially expressed transcripts by FDR were used to create heatmaps of significant DEGs. Pathway analysis was conducted using PathfindR (28 (link)). Library preparation and sequencing was conducted by the Whitehead Institute Genome Technology Core (Boston, MA).
10
Transcriptomic Profiling of Mouse Samples
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RNA concentration was estimated using the Agilent High Sensitivity RNA Screen Tape System, and libraries were prepared using the SMART-Seq® v4 Ultra® Low Input Kit (Takara Bio) with 5 ng total RNA input per sample, as per manufacturer recommendations. cDNA libraries were subject to high-throughput sequencing (Illumina NovaSeq) and ~50 million paired-end reads were generated per sample. Reads were checked for quality (FastQC v0.11.5) and processed using the Digital Expression Explorer 2 (DEE2) workflow55 . Adapter trimming was performed with Skewer (v0.2.2)56 (link). Further quality control was performed with Minion, part of the Kraken package57 (link). Filtered reads were mapped to the mouse reference genome GRCm38 using STAR aligner58 (link) and gene-wise expression counts were generated using the “-quantMode GeneCounts” parameter. The R package edgeR was used to calculate FPKM59 (link).
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