title: GSM6722395: Single oocyte (DRaqL - SMART-Seq v4 3' DE Kit) DpOo01_10_S7_IL10; Mus musculus; RNA-Seq
library_construction_protocol: A Smart-seq v4 3' DE Kit (Takara Bio) was adapted to DRaqL as follows.  Cells were isolated with LCM in 6.4 μL of cell lysis buffer (0.25 μL of 40 U/μL Takara RNase Inhibitor, 0.4 μL of 100 mM DTT, 0.16 μL of 1:500,000 diluted ERCC RNA Spike-In Mix 1, 0.4 μL of 5% SDc, and 5.19 μL of nuclease-free water) in the caps of PCR tubes, and spun down into the tubes by brief centrifugation.  Cells were then lysed at 72°C for 3 min, followed by the addition of 2.8 μL quenching buffer (0.7 μL of Triton X-100, 0.7 μL of 2% BSA, and 1.4 μL of 5× SuperScript II buffer [Thermo Fisher]) to reduce the denaturing effect of SDc.  Then, 2.8 μL of RT oligo (1 μL of 12 μM Oligo dT In-line Primer and 1.8 uL of nuclease-free water: kit components) was added, followed by incubation at 72°C for 90 sec.  Reverse transcription was performed by adding 7.5 μL of Master Mix (4 ?L of 5× Ultra Low First-Strand Buffer, 1 μL of 48 μM SMART-Seq V4 oligonucleotide, 0.5 μL of 40 U/μL RNase Inhibitor, and 2 μL of SMARTScribe Reverse Transcriptase: kit component) supplemented with 0.25 μL of SuperScript II and 0.25 μL of SuperScript III, and by incubating at 42°C for 90 min and 70°C for 10 min.  Then, cDNA amplification was performed by adding 30 μL of kit component PCR Mix (25 μL of 2× SeqAmp PCR Buffer, 1 μL of 12 μM Blocked PCR Primer II A, 1 μL of SeqAmp DNA polymerase, and 3μL of nuclease-free water) and applying the thermal cycling program for SMART-seq v4.  The amplified cDNA (50 μL) was purified with a 0.8× volume of AxyPrep MAG PCR clean-up reagent according to the manufacturer's instructions. The cDNA synthesis and amplification onward were performed as described previously with minor modifications [Kurimoto, K.et.al., Nucleic Acids Research, 34(5), e42.]. In brief, the first-strand cDNA was synthesized by SuperScript III [Thermo Fisher] and incubating at 50℃  for 5 min followed by heat inactivation at 70℃  for 10 min.  The excess RT primers were degraded by exonuclease I and incubating at 37℃ for 30 min followed by heat inactivation at 80℃ for 25 min. The first-strand cDNA product was poly dA-tailed by TdT and incubating at 37℃  for 1 min followed by heat inactivation at 70℃ for 10 min. Second-strand cDNA synthesis were performed by adding 9.5 μL reaction mixture of ExTaq, Hot Start version [Takara Bio] and V3[dT]24 primer in the ExTaq buffer with the following thermal cycling program (95℃  for 3 min, 50℃  for 2 min, and 72℃  for 3 min). cDNA amplification was performed by addingthe mixture of V1[dT]24 and ExTaq, Hot Start version [Takara Bio] in the ExTaq buffer with the following thermal cycling program (95℃  for 1 min; 20 cycles of 95℃  for 30 sec, 67℃  for 1 min and 72℃  for 3 min with a 6 sec elongation per cycle; and 72℃  for 10 min).  The amplified cDNA was purified with a 0.6× volume of AxyPrep MAG PCR clean-up reagent (Axygen) according to the manufacturer's instructions. Preparation of the libraries for SC3-seq was performed essentially as previously described (Nakamura et al., 2015)(Nakamura et al., 2017).  For Y-adapter sequencing, single-cell cDNAs were pre-amplified using the primers (V3[dT]24, V1[dT]24) and ExTaq HS with the thermal cycling program (95℃  for 3 min; 4 cycles of 95°C for 30 sec, 67℃  for 1 min and 72℃  for 5 min; 72℃  for 10 min). Pre-amplified cDNAs were then subjected to the primer-dimer removal, sonication, end polishing, and the size selection as in SC3-seq. Then, purified cDNA was dA-tailed in a 10 μL dA tailing mixture with incubation at 72℃  for 10 min. The dA-tailed product was subjected to the Y-shaped adaptor ligation by adding a mixture of Y-adaptor and NEB Next Quick Ligation Reaction Buffer and incubating at 20℃  for 15 min and at 72℃  for 20 min. The ligation product was purified with a 0.9×volume of AxyPrep reagent, and subjected to additional PCR amplification in 20 μL with the thermal cycling program (95℃  for 3 min, 55℃  for 1 min, and 72℃  for 1 min; 11 cycles of 95℃  for 30 sec, 60℃  for 1 min, and 72℃  for 1 min; and 72℃  for 3 min), followed by DNA purification with a 0.9×volume of AxyPrep reagent.