GSM4437351: HeLa_siRRP40_rep1_TIFseq; Homo sapiens; OTHER
We took 2.5 µg total RNA per sample as input. Input RNA was incubated with Turbo Dnase to prevent genomic DNA contamination. 5' phosphomonoesters of RNA degradation intermediate was dephosphorylated by Alkaline Phosphatase, Calf Intestinal. The 5'Cap of mature RNA was removed by incubating with Cap-Clip(0.125U/µl)  to allow for ligation to RNA adaptor. Mature RNA was ligated overnight with single strand DNA_RNA chimeric adaptor and was purified with 1.8X Ampure XP beads. Purified mature RNA worked as the template for reverse transcription(RT) to generate the first-strand cDNA with custom barcoded oligo dT primers. RNA template was then removed by RNase H and RNase cocktail mixture. The first-strand cDNA then worked as the template for PCR amplification with TERA(Takara). PCR products from different samples are then pooled together with equal mass. The pooled PCR products were then subjected to NOT1 HF (NEB) endonuclease digestion. To favour intramolecular ligation, the PCR products with sticky ends were highly diluted to a final concentration less than 1ng/µl and ligated with a high concentration of T4 DNA ligase at 16 ˚C for at least 16 hours. To remove the unligated linear PRC products, plasmid-safe were added. The self-circularized cDNA was purified with phenol-chloroform. Circularized cDNA was fragmented by Covaris ME220 sonicator (Duration 240s, peak power 30, duty factor 10, Avg Power(W) 6). The fragments were then purified with 1X Ampure XP beads according to manufacturer instruction. As the fragment of 5' and 3' docking site are labelled with biotin, these specific fragments are selected via streptavidin beads by incubating with M280 streptavidin beads. The fragments of docking site were then subjected to end repair, dA tailing and ligation of the barcoded partially forked adaptor with a common sequence to anneal to each other and the grafting part to anchor to Illumina sequencer flowcells. After final amplification using Phusion High Fidelity Master Mix with PCR graft P5 and P7 as primers for 11 cycles, the library was ready to load onto the sequencer. The library is ready to load onto sequencer after purification with 1X Ampure beads according to manufacturer instruction. We used four custom sequencing oligos. We added 15Ts to illumine read1 sequencing oligo and named it as SeqR1+15T. The sequencing oligos for index1, index2 and read2(SeqR2) are exactly the same as Illumina oligos. We diluted the oligos according to manufacturer instruction. We made two mixtures of sequencing oligos. Mixture 1 was composed of SeqR1+15T and SeqR2 in a 1:1 ratio. Mixture 2 was composed of index1 and index2 sequencing primer in 1:1 ratio. The mixture 1 was loaded into both positions for both custom read1 and read2 primers on Illumina sequencing kit. The mixture2 was loaded on custom index primer position on sequencing kit. TIFSEQ2 library was denatured, diluted and loaded according to manufacturer instruction. We used the stand-alone configuration of the NextSeq 500 sequencing instrument. We checked read1, index1, index2 and read2 as custom sequencing oligos. For paired-end sequencing, we set as read1 76bp, read2 76bp, index1 6bp, index2 6bp. RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions.
GEO Accession
GSM4437351