TSS-seq of Vv1724
Yeast cell cultures were grown in liquid YPD media and cells were harvested at the mid-log phase. The library construction for TSS-seq essentially as described by Vvedenskaya et al., 2015; the steps are briefly described as follows; 100 g of the extracted total RNA (phenol-chloroform extraction) was treated with 30 U of DNase I (QIAGEN) and purified using RNeasy Mini Kit (QIAGEN). A riboPOOL probe (siTOOLS Biotech) was used to deplete rRNAs from DNase-treated RNAs. rRNA depletion was done following published riboPOOL Protocol_v1-6 from siTOOL Biotech which was modified by us to deplete 25ug of DNAse-treated RNAs at once. The rRNA-depleted RNA was resuspended in ~15 l of nuclease-free water. From rRNA-depleted RNA, to remove RNA transcripts carrying a 5 monophosphate moiety (5-P), ~1 g of rRNA-depleted RNA were treated with 1 U Terminator 5-Phosphate-Dependent Exonuclease (Epicentre) in the 1 Buffer A in the presence of 40 U RNaseOUT in a 50 l reaction at 30C for 1 hr. Then, RNA was extracted using the phenol-chloroform method. Next, to get rid of 5-terminal phosphates, RNAs were treated with 1.5 U CIP (NEB) in 1 NEBuffer three in the presence of 40 U RNaseOUT in a 50 l reaction at 37C for 30 min. After the reaction was completed, RNAs were again extracted using the phenol-chloroform method. Now to convert enriched  5-capped RNA transcripts to 5-monophosphate RNAs, CIP-treated RNAs were mixed with 12.5 U CapClip (Cellscript) and 40 U RNaseOUT in 1 CapClip reaction buffer in a 40 l total volume and incubated at 37C for 1 hr. Then, the reaction went through the phenol-chloroform extraction method. To ligate the 5 adapter, these CapClip-treated RNA products were mixed with 1 M 5 adapter oligonucleotide s1086, 1 T4 RNA ligase buffer, 40 U RNaseOUT, 1 mM ATP, 10% PEG 8000, and 10 U T4 RNA ligase 1 in a 30 l reaction volume. The reaction was incubated at 16C for 16 hr and was halted by adding 30 l of 2 RNA loading dye. The mixtures were then separated by using electrophoresis on 10% 7 M urea slab gels in 1 TBE buffer. Then the gel was incubated with SYBR Gold nucleic acid gel stain. RNA products migrating above the 5 adapter oligo were recovered from the gel as described (Pinto et al., 1994),  and were purified by ethanol precipitation. To generate the first strand cDNA, 5-adaptor-ligated products were mixed with 0.3 l of 100 M s1082 oligonucleotide containing a randomized 9 nt sequence, incubated at 65C for 5 min, and cooled to 4C. A mixture of 4 l of 5 First-Strand buffer, 1 l (40 U) RNaseOUT, 1 l of 10 mM dNTP mix, 1 l of 100 mM DTT, 1 l (200 U) of SuperScript III Reverse Transcriptase and 1.7 l of nuclease-free water was added to the reaction. The reaction was incubated at 25C for 5 min, 55C for 60 min, 70C for 15 min, and then cooled to 25C. 10 U RNase H was added, the mixtures were again incubated for 20 min at 37C, and 20 l of 2 DNA loading solution (PippinPrep Reagent Kit, Sage Science) was added. Then, the DNAs were separated by electrophoresis on 2% agarose gel (PippinPrep Reagent Kit, external Marker B) to collect a library of ~90 to ~ 550 nt range which is recovered by ethanol precipitation. To amplify cDNA, 9 l of gel-isolated cDNA was added to the mixture containing 1 Phusion HF reaction buffer, 0.2 mM dNTPs, 0.25 M of one of the i5 index containing lab-designed custom primer (CKO3414-3423), 0.25 M of one of the Illumina index primer (RPI1-32) and 0.02 U/l Phusion HF polymerase in 30 l reaction. Finally, PCR-amplified libraries were isolated by electrophoresis on 2% agarose gel and products in the range of ~180 to ~ 550 nt were collected following ethanol precipitation. Thus, generated dual indexed TSS-seq libraries which were sequenced together with STRIPE-seq libraries. TSS-seq and STRIPE-seq libraries were pooled and sequenced on an Illumina NovaSeq-S1-200 cycles sequencing flowcell using custom sequencing primer.