Human PBMCs and blister cells were stained with antibodies in PBS for 10 min, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 µl of 0.2% Triton plus 1 U/µl RNaseOUT (Thermo Fisher Scientific). 182 blood cells and 273 skin cells were collected, and single-cell libraries were prepared using the Smart-Seq2 protocol (Picelli et al., 2014 (link)) with minor modifications. We performed reverse transcription using Smartscribe RT enzyme (50 U/cell; Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying quality control (QC) filters.
Smartscribe rt enzyme
Manufactured by Takara Bio
Smartscribe RT enzyme is a reverse transcriptase enzyme used in the synthesis of complementary DNA (cDNA) from RNA templates. It possesses RNA-dependent DNA polymerase activity, allowing for the conversion of RNA into single-stranded cDNA.
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Lab products found in correlation
2 protocols using smartscribe rt enzyme
1
Single-cell Transcriptomics of PBMCs and Skin Cells
2
Single-cell transcriptome profiling
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Following FACS sorting of single cells into lysis buffer, mRNA was primed in each well with an oligo dT primer that incorporates a unique 10 base pair (bp) barcode and Takara's SMART sequence to the 3′ ends and reverse transcribed to yield cDNA using SMARTscribe RT enzyme (Takara) and a template switching oligo to incorporate the SMART sequence to the 5′ ends. These cDNAs from each well were combined and purified with Ampure XP beads and amplified for 16 cycles using an oligo corresponding to the SMART sequence. Full-length cDNAs were sheared using Covaris E220 instrument with peak incident power 18, 20% duty factor, 50 cycles/burst for 2 min. The cDNAs were blunt-ended, an A base added to the 3′ ends, and Illumina sequencing adapters ligated to these ends. Ligated fragments were then amplified for 16 cycles using standard i7 indexing primer and P5 primer that binds only to the 3′ ends of the cDNAs containing SMART sequences and barcodes. Fragments were sequenced on an Illumina HiSeq-2500 using paired reads extending 26 bases for read one to read cell barcodes, and 98 bases for read two for mRNA data. Each plate was represented by a unique index and each well is represented by unique barcode.
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