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Briefly, as our previous studies described, the cell lysis buffer mixture containing 4 U RNase inhibitor (TAKARA, Cat. 2313B), 0.25% IGEPAL CA-630 (SIGMA, Cat. I3021), and in vitro methylation mix (NEB, Cat. M0227L) were prepared for an individual cell. Then the DNA and RNA of single cells were isolated by a nuclear separation mixture containing 0.2µl of Dynabeads Myone Carboxylic Acid (Invitrogen, Cat. 65011), 0.2% Tween-20 (SIGMA, Cat. P1379), 1% Triton X-100 (SIGMA, Cat. T8787), 4 U RNase inhibitor, 50mM DTT and 2µl of 5X Superscript II first-strand buffer (Invitrogen, Cat. 18064071). Subsequently, for RNA part, single cell RNA-seq library was constructed following modified Smart-seq2 protocol. The high-quality libraries were sequenced with 50bp pair-end reads on Illumina Hiseq Xten (Novogene) |
RNA-Seq |
TRANSCRIPTOMIC SINGLE CELL |
PolyA |
PAIRED
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