RNA-Seq of HeLa cells
TRIzol reagent (Invitrogen) was used to isolate total RNA from HeLa cells and NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, cat. no. E7775L) was used to prepare the cDNA libraries. In brief, dilute 1 g qualified DNA-free total RNA with nuclease-free water to a final volume of 50 L. Add 50 L actived RNA capture beads to each RNA sample and mix thoroughly. Place the tube in a thermocycler and heat the sample at 65C for 5 minutes and cool to 4C. Incubate the sample at RT for 5 minutes to allow the mRNA to bind to the beads. Then using magnetic rack to isolate and purify the mRNA. To elute the mRNA from the beads and fragment, add 11.5 l of the first strand synthesis reaction buffer and random primer mix (2X), pipette up and down to resuspend the beads. Incubate the sample in a thermocycler and heat the sample 15 minutes at 94C and hold at 4C. Quickly spin down the tube in a microcentrifuge to collect the liquid from the sides of the tube and place on the magnet right away and wait 5 mins for the solution to be clear. Collect the fragmented mRNA by transferring 10 L of the supernatant to a nuclease-free 0.2 ml PCR tube. 10 L fragmented mRNA, 8 L nuclease-free water and 2 L first strand synthesis enzyme mix were mixed and placed in the thermocycler (25C for 10 minutes, 42C for 15 minutes, 70C for 15 minutes, hold at 4C) to synthesize the first strand cDNA. Assemble the second strand cDNA synthesis reaction on ice by adding first strand cDNA 20 L,8 L reaction buffer (10X), 4 L enzyme mix and 48 L nuclease-free water. Mix the mixture thoroughly by pipetting the reaction up and down and incubate in a thermocycler for 1 hour at 16C. Then purified the double-stranded cDNA using SPRIselect beads and eluted in 50 L TE buffer. 50 L double-stranded cDNA, 7 L reaction buffer and 3 L enzyme mix were mixed and incubated in a thermocycler ( 20C for 30 minutes, 65C for 30 minutes, and hold at 4C). Assemble the ligation reaction on ice by adding the 60 L end prepped DNA, 2.5 L diluted adaptor, 1 L ligation enhancer and 30 L ligation master mix, and incubate 15 minutes at 20C in a thermocycler, and then, add 3 L USER enzyme to the ligation mixture and incubate at 37C for 15 minutes. Purification of the ligation reaction using SPRIselect beads. Then, performed PCR enrichment of adaptor ligated DNA with the component of 15 L adaptor ligated DNA, 25 L Master Mix, 5 L i5/i7 primer. Place the tubes on a thermocycle with a heated lid using the following cycling conditions: 98 C for 30 s; 12 cycles of 98 C for 10 s and 65 C for 95 s; final extension at 65 C for 5 min and hold at 4 C. Purification of the PCR reaction using SPRIselect beads. qPCR was used to measure the concentration of each sample and Fragment Analysis capillary electrophoresis was used to determine the size distribution of libraries, using 2 L following the manufacturers instructions. Run a bead selection to choose the DNA fragment size from 300 to 500 bp using Ampure XP beads. Then the library was pooled together with equal molar and perform paired-end sequencing on a single lane of an Illumina NovaSeq system yielding 150-bp paired-end (PE150) reads.