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Intestinal stem cells were cultured using established protocols and harvested upon reaching ideal growth. Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen). Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. 1 μg total RNA was used for following library preparation using a VAHTS mRNA-seq V3 Library Prep Kit for Illumina(NR611). The poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second-strand cDNA were synthesized. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Then libraries with different indexs were multiplexed and loaded on an Illumina Novaseq 6000 instrument for sequencing using a 2x150 paired-end (PE) configuration according to manufacturer’s instructions. |
RNA-Seq |
TRANSCRIPTOMIC SINGLE CELL |
PolyA |
PAIRED
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