Instrument: AB 5500xl Genetic Analyzer
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Total RNAs from the mouse embryonic stem cell (mESC) line BVSC R8 were extracted using an RNeasy mini kit [Qiagen (74104), Hilden, Germany] according to the manufacturer’s instructions. The isolated RNAs were serially diluted by double-distilled water (DDW) to concentrations of 250 ng/μl, 25 ng/μl, 2.5 ng/μl, 250 pg/μl, and 25 pg/μl for use in evaluation of the quantitative performance of the SC3-seq. For isolating mouse blastocysts, C57BL/6 mice were mated and noon of the day when a copulation plug was identified was designated as embryonic day (E) 0.5. At E4.5, peri-implantation blastocysts were flushed from the uteri by KSOM [Merck Millipore (MR-020P-5D), Darmstadt, Germany], and then they were bisected into a polar part containing an inner cell mass (ICM) and polar trophectoderm (pTE) and a mural part containing mural TE (mTE) by a glass needle under a dissection microscope [Leica Microsystems (M80), Wetzlar, Germany]. Each fragment was incubated with 0.25% trypsin/PBS [Sigma-Aldrich (T4799), St. Louis, MO] for around 10 min at 37°C, then dissociated into single cells by repeated pipetting, and dispersed in 0.1 mg/ml of PVA/PBS [Sigma-Aldrich (P8136)] in preparation for the SC3-seq analysis. For the isolation of single hiPSCs cultured with the feeders, the culture was first treated with CTK solutions [0.25% Trypsin [Life Technologies (15090-046)], 0.1 mg/mL Collagenase IV [Life Technologies (17104-019)], 1 mM CaCl2 [Nacalai Tesque (06729-55)] (Fujioka, T., et al. (2004). Int.J.Dev.Biol., 48(10), 1149.)] for the removal of the feeder cells, then dissociated into single cells using Accutase [Innovative Cell Technologies, San Diego, CA]. For the preparation of single cells from a feeder-free system, the cells were dissociated into single cells with 0.5 × TrypLE Select [TrypLE Select [Life Technologies (12563011)] diluted 1:1 with 0.5 mM EDTA/PBS] [Nakagawa, M., et al. (2014). Scientific Reports, 4, 3594.]. Dissociated single hiPSCs were transferred several times into a pick-up medium consisting of 1% KSR/PBS containing 10 μM of the ROCK inhibitor Y-27632 [Wako Pure Chemical Industries (257-00511)] [Watanabe, K., et al. (2007). Nature Biotechnology, 25(6), 681–686.] in preparation for the SC3-seq analysis. cDNA synthesis and amplification from isolated RNAs/single cells were perfomed essentially as reported in [Kurimoto, K.et.al., Nucleic Acids Research, 34(5), e42.] except that the spike-in RNAs developed by the External RNA Control consortium [ERCC; Life Technologies (4456740)] were used and different numbers of PCR cycles were employed for amplification depending on the amounts of starting total RNAs (total RNA 100 ng: 7 cycles; 10 ng: 11 cycles; 1 ng: 14 cycles; 100 pg: 17 cycles; 10 pg: 20 cycles). 5 ng of amplified and quality-checked cDNAs were added to the pre-amplification buffer [1 × ExTaq buffer [Takara Bio (RR006), Shiga, Japan], 0.2 μM of each dNTP [Takara Bio (RR006)], 0.01 μg/μl of the N-V3 (dT)24 primer (HPLC-purified, attachment of amine at the 5-prime end), 0.01 μg/μl of the V1(dT)24 primer (HPLC-purified), and 0.025 U/μl of ExTaqHS [Takara Bio (RR006)]], and were amplified by four cycles of PCR. The surplus primers and PCR byproducts such as primer dimers were removed by size selection through three rounds of purification using a 0.6 × volume of AMPureXP beads for each round [Beckman Coulter (A63881), Tokyo, Japan] according to the manufacturer’s instructions. The purified cDNAs were diluted to 130 μl by double-distilled water (DDW) and fragmented by shearing with Covaris S2 or E210 (duty cycle: 10%; intensity: 5; cycles per burst: 200; duration: 50 seconds; an intensifier was installed in the case of E210) [Covaris, Woburn, MA], and then end-polished in the End-polish buffer [1 × NEBnext End Repair Reaction buffer [NEB (B6052S), Ipswich, MA], 0.01 U/μl of T4 DNA polymerase [NEB (M0203)], and 0.033 U/μl of T4 polynucleotide kinase [NEB (M0201)]] for 30 min at 20oC. After incubation, a 0.8 × volume of the AMPureXP was immediately added, the solution was mixed for more than 20 min, and then the supernatant was transferred to a 1.2 × volume of the AMPureXP reagent and the cDNAs were purified. Next, to provide the purified cDNAs with an Int-adaptor sequence, the cDNAs were incubated in 30 μl of the Internal adaptor extension buffer [1 × ExTaq Buffer, 0.23 mM of each dNTP, 0.67 μM of the IntV1 (dT)24 primer (HPLC-purified), 0.033 U/μl of ExTaqHS] using the following thermal cycler program: 95oC for 3 min, 67oC for 2 min, and 72oC for 2 min. The reactions were terminated by chilling in an ice-block, and after the addition of 20 μl of the P1-adaptor ligation buffer [a mixture of 10 μl of 5 × NEBNext Quick Ligation Reaction Buffer [NEB (B6058S)], 0.6 μl of 5 μM of the P1-T adaptor [Life Technologies (4464411)], and 1 μl of T4 ligase [NEB (M0202M)]], the solution was incubated for 15 min at 20oC and for 20 min at 72oC. After two rounds of cDNA purification by adding a 1.2 × volume of AMPure XP, the cDNAs were added into the Final amplification buffer [1 × ExTaq buffer, 0.2 mM of each dNTP, 1 μM of the P1 primer, 1 μM of the BarT0XX_IntV1 primer (HPLC-purified), 0.025 U/μl of ExTaqHS] and amplified by PCR using the following thermal cycler program: 95oC for 3 min; followed by 9 cycles of 95oC for 30 sec, 67oC for 1 min and 72oC for 1 min; with a final extension of 72oC for 3 min. Finally, the cDNA libraries were purified two times by using a 1.2 × volume of AMPureXP and dissolved in 20 μl of TE buffer. The quality and quantity of the constructed libraries were evaluated by LabChip GX or Bioanalyzer 2100, a Qubit dsDNA HS assay kit [Life Technologies (Q32851)], and a SOLiD Library TaqMan Quantitation kit [Life Technologies (4449639)]. The cDNA libraries were sequenced with an ABI SOLiD 5500XL system (Applied Biosystems) for 50-bp single-end reads according to the instructions of the manufacturer.