Qiaseq fx single cell rna library kit

SMART-Seq HT Kit (Takara Bio), named as method X, is a PCR-based method that uses oligo-dT primers and template switching. LNA technology is used in this method for efficient cDNA synthesis by template switching oligonucleotides containing modified guanosine and locks the first-strand cDNA. NEBNext Single Cell/Low Input RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA), named as method Y, is a PCR-based method that uses oligo-dT primers and template switching only. QIAseq FX Single Cell RNA Library Kit (Qiagen), named as method Z, is an MDA-based method that uses oligo-dT primers and phi29 DNA polymerase. Experimental procedures were performed by well-disciplined technicians according to the manufactures’ instructions.

Additional rRNA removal step was performed on n = 6 DNase treated RNA (two for each kit). The rRNA removal was performed using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs, Ipswich, MA) as per the manufacturer’s instructions. Each viral extract was then subjected to Whole Transcriptome Amplification (WTA) using the QIAseq FX Single Cell RNA Library Kit (Qiagen, Hilden, Germany) as per manufacturer’s instructions to generate the cDNA. Amplified cDNA was quantified using the Qubit® DNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA) with the Qubit® 3.0 Fluorometer.

Libraries were prepared using the QIASeq FX Single Cell RNA Library Kit (Qiagen) according to the manufacturer's instructions. The DNA libraries were generated and analysed for average fragment size distribution using the Agilent 2100 Bioanalyzer (Agilent Technologies). Metagenomic sequencing was performed on the Illumina MiSeq system with the reagent kit v3 (Illumina) for 600 cycles to generate 2 × 250 bp paired‐end reads.

For RNA extraction, samples were thawed on ice. They were then washed with cold PBS and spun down in a centrifuge for 5 min at 1000 rpm to remove the buffer; this process was repeated 3 times. Cell lysis, cDNA conversion, fragmentation, and library preparation were done in single tubes using the QIAseq FX Single Cell RNA Library Kit (Qiagen). Final library amplification was performed using Q5 Hot Start polymerase (New England Biolabs), utilizing indexing primers and universal Illumina adapters obtained from the Functional Genomics Lab (FGL) at the University of California, Berkeley. Final libraries, which were constructed for each individual sample, were submitted to the FGL for library quality check via Bioanalyzer (Agilent) and subsequent 150 bp paired-end sequencing on Illumina NovaSeq 6000 S4 flow cells, attempting 37 million reads per library.

Total RNA was extracted from primary mouse hair cells (at postnatal day 1), mouse iHCs (at day 14 post infection with reprogramming factors) and MEFs (at 14 days post transduction with dsRed). For each replicate 20,000 FACS-sorted cells were used as input for RNA-seq. Total RNA was extracted with either Quick-RNA Microprep kit (Zymo Research), quantified by bioanalyzer and then processed for libraries with either QIAseq FX Single Cell RNA Library Kit (Qiagen) or TruSeq RNA Library Prep Kit v2 (Illumina). Specific sequencing parameters and instrument models were submitted with GEO datasets. At least three replicates were collected for each condition and sequenced to a depth of at least 20 million reads.
Reads were mapped to the mouse reference genome (Gencode Mm10v11) using STAR. Read counts were quantified by RSEM. Only protein coding polyA tail transcripts and autosomal genes were kept. Transcript counts were collapsed to gene counts. Differentially expressed genes were identified using the DESeq2 package. Genes with a log fold change threshold greater than one and adjusted P-value of less than 0.1 were considered significant. Principle component analysis and unsupervised hierarchical clustering of RNA-seq was performed using counts transformed by DESeq2’s regularized logarithm (Rlog).
GEO accession number: GSE149260.

About 800 SCs from mice (homeostatic condition or 48 h-post injury) were isolated by FACS, and the mRNA library was constructed using the QIAseq FX Single Cell RNA Library Kit (QIAGEN) according to the manufacturer’s instruction. Libraries were sequenced by the Illumina HiSeq 2000 platform as 150-bp pair-ended reads. Reads were aligned using bowtie v0.12.9. FPKM estimation was performed with Cufflinks v2.1.1, aligned reads were counted with HTSeq, and differential expression analysis was performed with DESeq2. Differentially expressed genes were selected using a cut-off at a p value of <0.05 (false discovery rate adjusted for multiple testing).