GSM5769570: GS689-3 (LRCA); Homo sapiens; RNA-Seq
Total RNA from GS689 cells were extracted using TRIzol reagent (Invitrogen, #15596018) 500 ng of total RNA extracted from GS689 cells were used as templates for cDNA synthesis following the R2C2 method with some modifications. The reverse transcription and template-switching reaction was performed by Maxima H minus reverse transcriptase (Thermo Scientific, #EP0751) under the following conditions: 42°C for 90 min, 85°C for 5 min. Additional RT oligos were digested using 1 μl of Exonuclease I at 37°C for 20 min and 80°C for 10 min. After reverse transcription, a UMI sequence was added to the 3' end of cDNA by strand extension reaction using Phusion HiFi DNA polymerase (NEB, #M0530), a template oligo including 60 random bases of C, G, and T (B60), and a 3' 3C-spacer. The reaction was performed by incubating at 95°C for 3 min, followed by 5 cycles of (98°C for 15 s, 67°C for 15 s, 72°C for 15 s) with a final extension at 72°C for 5 min. UMI-coded cDNA products were aliquoted into multiple tubes and PCR amplified using KAPA HiFi ReadyMix by incubating at 95°C for 3 min, followed by 13 cycles of (98°C for 20 s, 67°C for 15 s, 72°C for 4 min) with a final extension at 72°C for 5 min. PCR products were purified with 1X volume of SPRIselect beads. A ~100 bp dsDNA sequence was amplified from Lambda DNA as a DNA splint for circularization of cDNA. The PCR conditions of DNA splint were the same as that of cDNA except that 30-cycle PCR and 20 s of extension step in each cycle were executed. Equal amounts of cDNA and DNA splints (~200 ng) were used for Gibson Assembly reaction by incubating with 2X NEBuilder HiFi DNA Assembly Master Mix (NEB, #E2621) at 50°C for 60 min. Linear DNA was digested using a mixture of Exonuclease I and III (NEB, #0206) at 37°C for 4-12 hrs and 80°C for 15 min. A universal oligo that anneals to the PCR adapter sequence of circular dsDNA was used to initiate linear rolling circular amplification. The reaction was performed using 1 μl of Phi29 DNA polymerase at 30°C for 16 hrs and 65°C for 15 min. LRCA products were purified with 0.7X volume of SPRIselect beads. The second strand synthesis of LRCA-generated ssDNA products was performed using Phusion DNA polymerase and Taq DNA ligase (NEB, #M0208) by gap-filling and ligation reaction at 50°C for 60 min. dsDNA sequences were then sheared by g-TUBE (Covaris, #520079) to the desired size (around 8-10 kb) following the vendor's instructions. Sheared DNA products were purified with 0.5X volume of SPRIselect beads and subjected to 1D library construction following the standard SQK-LSK109 protocol. cDNA products were end-repaired and dA-tailed using NEBNext Ultra II End Repair/dA-Tailing Module (NEB, # E7546) by incubating at 20°C for 20 minutes and 65°C for 20 min. End-prepared cDNA were purified with 1X volume of AMPure XP beads and eluted in 60 μl of Nuclease-free water. Adapter ligation was then performed using NEBNext Quick T4 DNA ligase (NEB, #E6056) at room temperature for 10 min. After ligation, the libraries were purified using 0.45X volume of AMPure XP beads and short fragment buffer (SFB) to enrich all fragments equally.