GSM8865328: mESC-TT_control_R1; Mus musculus; OTHER
RNA was extracted from yeast as previously detailed (Chia et al. 2021). For RNA extraction from yeast grown in YPD, 24 optical density (OD) units for TSS-seq were collected by centrifugation, and snap-frozen in liquid nitrogen. Per 10 OD units, RNA was extracted with 1 ml Tris-EDTA-SDS (TES) buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5% SDS) and 1 ml Acid Phenol:Chloroform:Isoamyl alcohol (125:24:1, Ambion), by shaking at 65°C for 45 minutes at 1400 rpm. After centrifugation at 4°C, max speed, 10 minutes, the aqueous phase was transferred to cold ethanol with 0.3 M sodium acetate. Precipitation was carried out overnight at 4°C, before centrifugation at 4°C, max speed, 20 minutes. The pellet was washed once with 80% v/v ethanol, dried, and reconstituted in DEPC-treated sterile water. rDNase (Machery-Nagel) treatment was carried out for 20 minutes at 37°C, before spin column purification (Machery-Nagel).  RNA was extracted from mammalian cells using TRIzol (Thermo Fisher). For TRIzol RNA extraction, 1 ml of TRIzol was added per 10-cm dish. Chloroform (0.2 volume, Thermo Fisher) was added, and the mixture was shaken for 30 seconds before centrifugation was performed. The aqueous phase was mixed with 1.1 volumes of isopropanol (Thermo Fisher), and RNA was precipitated overnight at 4°C. After centrifugation, the pellet was washed with 85% (v/v) ethanol, dried, and reconstituted in DEPC-treated sterile water. rDNase (Qiagen) digestion was carried out at 37°C for 1 hour before purification by spin column (Qiagen) or by phenol:chloroform and ethanol precipitation.  For small RNA size selection, Monarch RNA Cleanup with a 1:3 ratio of sample: ethanol volume was used.                                                                                                                     4SU RNA labelling 4SU labelling was carried out as detailed previously (Gregersen et al. 2020), with few modifications. Cell culture media was removed, and filtered, and 1 mM 4SU (Glentham Life Sciences) was added. The cells were returned to the incubator, and labelled with 4SU for 15 minutes before medium aspiration and TRIizol addition. 1/5 volume of chloroform was added, mixed, and then centrifuged at 12,000g for 15 minutes at 4°C. 1.1 volumes of isopropanol and 1 µl of glycoblue were mixed with the upper aqueous layer, precipitation then occurred overnight at -20°C. The pellet was washed once with 85% ethanol, dried, and resuspended.  RNA integrity was checked for a selection of samples using Bioanalyser (Agilent Technologies). 4SU incorporation was assessed using slot blot. Here, 5 µg of total RNA was mixed with 3 µl biotin buffer (833 mM Tris-HCl, pH 7.4, and 83.3 mM EDTA) and 50 µl of 0.1 mg/ml MTSEA biotin-XX linker (Biotium, dissolved in dimethylformamide; Sigma-Aldrich) to a total volume of 200 µl, and incubated in the dark for 30 minutes. RNA clean-up was performed by adding an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1 (vol/vol/vol)), mixing, and centrifuging at 12,000g for 5 minutes at 4°C. The aqueous phase was mixed with 1.1 volumes of isopropanol, 1/10 volume of 5 M NaCl and 1 µl of glycoblue and precipitated at -20°C for at least 2 hours. The pellet was washed once with 85% ethanol, dried, and resuspended to 10 µl. The TSS-seq protocol: total RNA was dephosphorylated with quickCIP (NEB, 1.2 U/µg RNA) 37°C, with RNasin Plus before heat inactivation. RNA was extracted using phenol/chloroform/isoamyl alcohol as before. RNA was treated with mRNA decapping enzyme (NEB, 0.7 U/µg RNA) at 37°C with RNasin Plus. RNA was extracted using phenol/chloroform/isoamyl alcohol as before. RNA and the 5' adaptor (10 µM) were denatured for 2 minutes at 70°C, then cooled on ice for 2 minutes. Ligation was performed, 16°C using and T4 RNA ligase I (30 U), with RNasin Plus. RNAClean XP (1.8X ratio, Beckman Coulter) was used to remove unligated adaptors, according to the manufacturer's instructions. The concentration of the samples was measured using Qubit RNA BR assay, and samples were multiplexed. polyA+ RNA was enriched using Dynabeads Oligo(dT)25 (Thermo Fisher). Yeast RNA was fragmented with alkaline fragmentation reagent (Ambion). RNeasy MinElute Clean-up columns, with 1.5x volume of Ethanol, were used to purify the samples. The 3' ends were fixed by CIP treatment (NEB,30 U).  Ligation was carried out by T4 Rnl2tr (NEB,200 U) for 2 hours, 25°C then 16 hours, 16°C. RNAClean XP (1.8X ratio) was used to remove unligated adaptors. RNA, RT oligo (0.5 pmol) and dNTP (10 mM each) were denatured at 65°C, then cooled on. Reverse transcription was carried out by SuperScript III (2 µl), with RNasin Plus with the following conditions: 25°C for 5 minutes, 42°C for 20 minutes, 50°C for 40 minutes, 80°C for 5 minutes. Template RNA was removed by RNase H (10 U) for 37°C, 30 minutes. AMPure XP beads (Agencourt, Beckman Coulter) were used to clean up the cDNA. 3X beads and 1.7x isopropanol added to the reverse transcription reaction, and incubated for 5 minutes. Beads were washed twice with 85% ethanol, dried, and eluted twice in nuclease-free water. cDNA pre-amplification was performed with i5_s and i7_s (300 nM each) primers and Phusion HF PCR Mastermix (Thermo Fisher). 6 PCR cycles were carried out (98°C for 10 seconds, 65°C for 30 seconds, 72°C for 30 seconds, with 3 minutes at 72°C final extension). ProNex beads (Promega) with a 1:2.95 ratio to sample were used to remove sequences less than 55 nt (including primer dimers). Final amplification was carried out with NEBNext i50 and i70 primers (500 nM each) and Phusion HF PCR Mastermix, for 8 cycles. Amplification was checked by using Novex 6% TBE (Tris/Borate/EDTA) gel and staining with SYBR green. ProNex beads (Promega) with a 1:2.3 ratio to sample were used for purification. Library concentration was measured using Qubit High Sensitivity D1000. Libraries were sequenced with Illumina NovaSeq 6000 using 100 nt paired-end reads, with 50 million reads minimum per sample.