title: GSM6722456: Single granulosa cell (DRaqL - Smart-seq2) SmSeq2DRaqL21_30_S7_IL04; Mus musculus; RNA-Seq
library_construction_protocol: Cells were isolated with LCM in 6.4 μL of cell lysis buffer (0.6 μL of 5× SuperScript II buffer, 0.1 μL of 100 μM Oligo dT VN, 0.8 μL of dNTP mix [25 mM each], 0.25 μL of 40 U/μL recombinant RNase inhibitor [Takara Bio], 0.5 μL of 100 mM DTT, 0.06 μL of 1 M MgCl2 [Sigma-Aldrich], 2 μL of 5 M Betaine [Sigma-Aldrich], 0.16 μL of 1:500,000 diluted ERCC RNA Spike-In Mix 1, 0.4 μL of 5% SDc, and 1.53 μL of DDW) in the caps of PCR tubes, and spun down into the tubes by brief centrifugation.  The isolated cells were lysed at 72°C for 6 min, followed by 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]).  Then, 1.6 μL of a template-switching mixture (0.1 μL of 100 μM N-template-switching oligo, 0.25 μL of SuperScript II, 0.25 μL of SuperScript III, 0.2 μL of recombinant RNase inhibitor, and 0.8 μL of DDW) was added, followed by the cycling RT program for DRaqL-Smart-seq2.  Then, cDNA amplification was performed by adding 15 μL of Seq amp PCR mixture (12.5 μL of 2x SeqAmp buffer (Takara Bio), 0.05 μL of 100 μM N-IS PCR primer, 0.5 μL of SeqAmp DNA polymerase (Takara Bio), and 1.95 μL of DDW) and applying the SeqAmp thermal cycling program.  For oocytes, we used a PCR program of 18 cycles.  The amplified cDNA (25.8 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.