GSM3661239: E_hep_3_01; Homo sapiens; RNA-Seq
Single-cell RNA-seq was performed according to the Smart-seq2 protocol as reported previously (Picelli et al., 2014). After the cells were digested into a single-cell suspension, each single-cell sample was manually selected under a microscope with a mouth pipette and transferred into lysis buffer containing 1.9 µl of 0.2% Triton X-100 (Sigma), 1 µl of 10 µM oligo (dT) primer, 1 µl of 10 mM dNTP mix (New England Biolabs), 0.1 µl of 40 U/µl RNase inhibitor (New England Biolabs) and 0.05 µl of ERCC spike-in RNA (a dilution of 1:300,000, Life Technologies). After single-cell lysis, the sample was mixed with 0.29 µl H2O, 2 µl of 5× Superscript II first-strand buffer, 2 µl of 5 M Betaine (Sigma), 0.5 µl of 100 mM DTT, 0.06 µl of 1 M MgCl2, 0.1 µl of 100 µM TSO primer, 0.25 µl of 40 U/µl RNase inhibitor and 0.5 µl of 200 U/µl SuperScript II reverse transcriptase (Invitrogen). After reverse transcription was performed, 2.25 µl H2O, 12.5 µl KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and 0.25 µl of 10 µM IS PCR primer were added to the sample. Then, PCR preamplification was conducted for 18 cycles. Next, cDNA samples were purified with VAHTS DNA Clean Beads XP beads (Vazyme). Purified cDNA (2 ng) was prepared for library construction using a TruePrep DNA Library Prep Kit (Vazyme) with a 0.4× standard reaction system, and PCR amplification was conducted for 8 cycles. Finally, the library was purified and size-selected, and fragments with a peak size of ~350 bp were analyzed with an Advanced Analytical Fragment Analyzer.
GEO Accession
GSM3661239