GSM9205084: ZC3H14_full_length_seq; Mus musculus; RNA-Seq
After digestion, single cells were placed into 2-μL lysis buffer by mouth pipette. The cell lysis buffer contained 2U RNase Inhibitor (Takara, Beijing, China, Cat. 2313B), 0.0475% Triton X-100 (Sigma-Aldrich, St. Louis, Missouri, USA, Cat. X100), 2.5-μM dNTP mixture (Thermo, Waltham, Massachusetts, USA, Cat. R0193), and 0.75-μM RT primer. We thoroughly vortexed the tubes for 60 seconds and incubated at 72°C for 3 minutes to release the linearized RNA molecules and immediately transferred them on ice. Then, 2.85 μL of RT mixture which comprised of 50U SuperScript II reverse transcriptase (Invitrogen, Cat. 18064071), 5U RNase Inhibitor, 5X Superscript II first-strand buffer, 5M betaine (Sigma-Aldrich, Cat. B0300), 25 mM DTT, 30 mM MgCl2 (Sigma-Aldrich, Cat. 63020), and 1.75-μM TSO primer was added into each tube. The RT reaction was carried out at 25°C for 5 minutes, 42°C for 60 minutes, 50°C for 30 minutes, and 70°C for 10 minutes. After that, 7.5-μL PCR mixture that included 2× KAPA HiFi Hot-Start Ready Mix and 300 nM of ISPCR oligo (AAGCAGTGGTATCAACGCAGAGT) was added into each tube. The amplification was performed by the following program: 4 cycles at 98°C for 20 seconds, 65°C for 30 seconds, and 72°C for 5 minutes, followed by 16 cycles at 98°C for 20 seconds, 67°C for 15 seconds, and 72°C for 5 minutes, with a final cycle at 72°C for 5 minutes. Then, we pooled the cDNAs of different cell barcodes together and purified twice with 0.6X Ampure XP beads (Beckman, Brea, California, USA, Cat. A63882). The concentration was measured using Qubit dsDNA HS and BR Assay Kits (Invi trogen, Cat. Q32854). The PCR product from about 60 blastomeres which were confirmed of effective amplification were pooled together in proportion to the number of amplified cycles. We took 100ng of the pooled cDNA tobuild PacBio sequencing library following the protocol of HIT-scISOseq and sequenced for 1 cell with HiFi mode.