title: GSM4460040: icSHAPE_siControl_Pulldown-rep1; Homo sapiens; OTHER
library_construction_protocol: . Forty 15-cm plates of HepG2 cells were prepared for 48-hour knockdown (siControl, siALKBH7, 5 plates per replicate). Upon the completion of knockdown, cells were collected by cell lifter in 5 mL cold DPBS and merged into one 50 mL centrifuge tube. The cell suspension was spun at 300g at 4 °C for 2 min to produce the cell pellet. For the cell pellet corresponding to one of four replicates in each case (4 of siControl or 4 of siALKBH7), add 500 uL control solution (450 uL DPBS + 50 uL DMSO) into two replicates of each case respectively, and add 500 uL NAI-N3 solution (450 uL DPBS + 50 uL 1.0 M NAI-N3 in DMSO) into other two replicates of each case respectively. Then incubate the resuspended solution at 37 ℃ for 5 min, followed by centrifuge at 300g for 2 min. Remove the supernatant carefully and wash the cell pellet with cold DPBS once, followed by centrifuge at 300g for 2 min again.   The cell pellet was resuspended with 1 mL NP-40 lysis buffer prepared in 10 mL volume: 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 10 mM MgCl2, 0.5% NP-40, plus 50 uL TURBO™ DNase (2 U/µL, AM2238, Invitrogen) and 500 uL SUPERase•In™ RNase Inhibitor (20 U/μL, AM2694, Invitrogen). Incubate the cell suspension at 4 °C for 30 minutes, followed by a spin at 16,000g at 4 °C for 5 min. The supernatant was transferred to a new Eppendorf tube and keep 10 uL of that as input with TRIzol added. The 1 mL lysate was dilute to 5 mL with IP wash buffer and then 250 uL of J2-Dynabeads was added for an incubation at 4 °C for 2 hours. The magnetic beads were washed once with 1 ml of IP wash buffer and transferred into a new Eppendorf tube by 500 µL of IP wash buffer. The beads were further washed twice by 1 mL of IP wash buffer, and J2-bound dsRNA was extracted with TRIzol reagent. Dilute the harvested dsRNA in 17 uL RNase-free water (for both DMSO-treated and NAI-N3 treated samples), and then add 2 uL of 2 mM sDIBO-biotin together with 1 uL SUPERase•In™ RNase Inhibitor. Incubate at 37 °C for 1 hour and purify RNA through Oligo Clean & Concentrator (Zymo Research). Dilute the purified RNA with 17 uL RNase-free water and add 2 uL RNA Fragmentaion Buffer (NEBNext® Magnesium RNA Fragmentation Module), followed by heating at 95 °C for 3 min. Put all samples onto ice immediately and add 2 uL Stop Buffer. RNA purification was performed for the 4 samples from DMSO treated case using Oligo Clean & Concentrator (Zymo Research), to afford “input” samples. For other 4 samples from NAI-N3 treated case, mix the RNA solution with 20 uL freshly-prepared C1 beads suspension (10 uL Dynabeads™ MyOne™ Streptavidin C1 beads washed twice with 200 uL 1X B&W buffer and stored in 20 uL 2X B&W buffer based on the manufacturer's protocol). After the incubation of 30 min at 4 °C, wash the beads three times with icSHAPE wash buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% NP40), followed by proteinase K digestion at 55 °C for 30 min. The harvested RNA (serving as “pulldown”, from NAI-N3 treated) was purified by Oligo Clean & Concentrator (Zymo Research), and sent for library construction together with “input” samples using SMARTer Stranded Total RNA-Seq Kit v2 (Takara). All libraries were sequenced on Illumina Illumina NovaSeq 6000 with single-end 100 bp read length.