GSM4200180
GSM4200180: Wild-type TIFseq premeiotic replicate 2; Saccharomyces cerevisiae; OTHER
Cell pellets were washed once with sterile water and snap-frozen in liquid nitrogen. Total RNA was extracted using hot acid phenol:chloroform:isoamyl alcohol (25:24:1) and TE-SDS buffer, then precipitated in ethanol with 0.3 M sodium acetate before re-suspension in RNAse-free sterile water. Briefly, we selected the capped RNA and used dT primer to generate cDNA. We then circularized the cDNA and broke them with sonication. The joined fragments, which consisted of the docking site of the 5' and 3' end of cDNA, were selected by streptavidin beads and used for library preparation.In detail, we took 2.5 µg total RNA per sample as input. To prevent genomic DNA contamination, input RNA was incubated with Turbo DNase(0.12U/µl)(Takara) for 20min at 37˚C. After inactivation of DNase, we filtered out the RNA degradation intermediate via dephosphorylating the exposed 5' phosphomonoesters by Alkaline Phosphatase, Calf Intestinal(0.3U/µl)(CIP from NEB). Then we performed two rounds of phenol-chloroform RNA extraction to remove CIP residues in the reaction system completely. The 5'Cap of mature RNA was later removed by incubating with Cap-Clip(0.125U/µl) at 37˚C for 60min. Cap-clip treatment exposed the 5' phosphomonoester of mature RNA and allowed for ligation to RNA adaptor in the following steps. After phenol-chloroform purification, mature RNA was then ligated overnight at 16˚C at 5'with single strand DNA_RNA chimeric adaptor, which introduced an 8 mer unique molecular identifier(UMI) and a common anchor sequence for subsequent PCR amplification.On the second day, the ligated RNA was purified with 1.8X Ampure XP beads according to the manufacturer instruction. Purified mature RNA worked as the template for reverse transcription(RT) to generate first-strand cDNA with custom barcoded oligo dT primers, which introduce the sequencing primer 1, 3'index, NOT1 endonuclease digestion site and a 3' universal anchor sequence for subsequent PCR reaction. For details of RT, RNA template was first mixed with primer and dNTPs. The mixture was denatured at 65˚C for 5min and then put on ice. After adding first strand buffer, Trehalose and Risosafe, the reaction system was first incubated at 42˚C for 2min, and then 2 µl of superscript II enzyme was added to each reaction. The RT system went through 42 ˚C for 50min, 50 ˚C for 30 min, 55 ˚C for 30min. Then the RT enzyme was inactivated at 70 ˚C for 15min. To remove the RNA template, 0.5ul RNase H(5U/µl) and 0.5µl RNase cocktail were added and incubated at 37 ˚C for 30min. First-strand cDNA was then purified with 2X Ampure XP beads according to manufacturer instruction.We stored half of cDNA as a backup. The left half was split into two as the template for following PCR amplification with TERA(Takara). First, denature at 98 ˚C for 2min, then 98 ˚C 20s, 60 ˚C 30s 68 ˚C 5min (+10s/cycle) for 16 cycles and final extension was performed at 72 ˚C for 5min. Pool the two PCR system for the same sample together and purify with 1X Ampure XP beads according to manufacturer instruction. The PCR products are then quantified with qubit dsDNA HS assay. 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 at 37˚C for one hour. After inactivation of NOT1 at 65 ˚C for 20 min, we used 1.8X Ampure XP beads to purify.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(NEB)(66.6U/µl) at 16 ˚C for at least 16 hours. To remove the unligated linear PRC products, for every 100 µl system, 0.5 µl plasmid-safe were added and incubated at 37 ˚C for 1h. After inactivation at 70 ˚C for 30min, 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 (Invitrogen) for 30 min. 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 18 cycles, the library was ready to load onto the sequencer. The final PCR parameter was as following, first, denature at 98 ˚C for 30s, then 98 ˚C 20s, 65 ˚C 30s, 72 ˚C 30s for 18 cycles and a final extension at 72 ˚C for 5min.The library is ready to load onto sequencer after purification with 1X Ampure beads according to manufacturer instruction. We used four custom sequencing oligoes. We added 15Ts to illumine read1 sequencing oligo and named it as SeqR1+15T. The sequencing oligoes for index1, index2 and read2(SeqR2) are exactly the same as Illumina oligoes. We diluted the oligoes according to manufacturer instruction. We made two mixtures of sequencing oligoes. 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 custom read1 and read2 primers on sequecing kit. The mixture2 was loaded on index primer on sequencing kit.TIF2 library was denatured, diluted and loaded according to manufacturer instruction.we used stand alone configuration of NextSeq 500 machine. We checked read1, index1, index2 and read2 as custom sequencing oligoes. For paired end, we set as read1 76bp, read2 76bp, index1 6bp, index2 6bp. TIF-Seq
GEO
GSM4200180
GEO Accession
GSM4200180