Single S-EGFP or W-EGFP cells were plated by FACS (SH800, Sony, Tokyo, Japan) into individual wells of a 384-plate (Piko PCR Plate, Thermo Fisher Scientific) pre-loaded with lysis buffer. The CEL-Seq2 protocol established by Hashimshony et al.37 (link) was used for RNA extraction and library preparation. Briefly, the RNA of each cell was reverse transcribed using CEL-Seq primers containing an anchored poly(T), a 6-bp unique molecular identifier, a 5’ Illumina adapter (San Diego, CA, USA), a cell-specific 6-bp barcode, and a T7 promoter (Supplementary Table 2 ). The External RNA Controls Consortium spike-ins (Thermo Fisher Scientific) were added to each preparation. After second-strand synthesis reaction, the double-stranded cDNAs were transcribed in vitro by T7 RNA polymerase. The synthesized RNAs were reverse transcribed using random primers with the 3’ Illumina adapter. Finally, the libraries were amplified by PCR (11 cycles). The pair-ended CEL-Seq2 libraries were sequenced by HiSeq 2500 (Illumina).
Piko pcr plate
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The Piko PCR Plate is a 96-well PCR plate designed for use in polymerase chain reaction (PCR) applications. The plate is made of polypropylene and is compatible with standard PCR thermal cyclers.
Automatically generated - may contain errors
2 protocols using piko pcr plate
1
Single-cell RNA-seq using CEL-Seq2
2
Single-cell RNA-seq using CEL-Seq2
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Single S-EGFP or W-EGFP cells were plated by FACS (SH800, Sony, Tokyo, Japan) into individual wells of a 384-plate (Piko PCR Plate, Thermo Fisher Scientific) pre-loaded with lysis buffer. The CEL-Seq2 protocol established by Hashimshony et al. was used for RNA extraction and library preparation 35 . Briefly, the RNA of each cell was reverse transcribed using CEL-Seq primers containing an anchored poly(T), a 6-bp unique molecular identifier (UMI), a 5' Illumina adapter (San Diego, CA, USA), a cell-specific 6-bp barcode, and a T7 promoter (Supplementary Table 2). The External RNA Controls Consortium (ERCC) spike-ins (Thermo Fisher Scientific) were added to each preparation. After second-strand synthesis reaction, the double-stranded cDNAs were transcribed in vitro by T7 RNA polymerase. The synthesized RNAs were reverse transcribed using random primers with the 3' Illumina adapter. Finally, the libraries were amplified by PCR (11 cycles). The pair-ended CEL-Seq2 libraries were sequenced by HiSeq 2500 (Illumina).
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