title: GSM3574533: SATU1_59_S11_R1_001; Homo sapiens; OTHER
library_construction_protocol: Placenta samples, stored snap fozen or in RNAlater (Thermo Fisher Scientific) in -80°C, were thawed and homogenized using the FastPrep®-24 5G Classic Instrument (2 x 30 s at 4.5 m/s) and Lysing Matrix D tubes  according to manufacturer's instructions (MP Biomedicals, Santa Ana, CA). Samples were lysed using 600 µl RTL buffer and 6 µl ß-mercaptoethanol on 20 mg of tissue homogenate, followed by RNA extraction using the RNeasy Mini Kit according to manufacturer's instructions (Qiagen, Valencia, CA). RNA concentrations were measured by the NanoDrop® 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific) and diluted in 10 ng/µl. The 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA)  was utilized for measurement of RNA integrity (RIN) values. For most samples, the RIN values ranged from 7 to 8.8, but samples with the lower range of 4 to 6.9 gave highly comparable results in TAC assay testing, and were included in the final assays. RNA samples were quantitated with the Qubit® 2.0 Fluorometer using Invitrogen RNA HS Assay Kit (5 to 100 ng) or Invitrogen RNA BR Assay Kit (20 to 1000 ng) according to manufacturer's instructions  (Thermo Fisher Scientific). Non-skirted low profile PCR Strip Tube Plate (Thermo Fisher) was used with domed cap strips (Thermo Fisher). 1 µl of total-RNA sample was used. The RIN values 7.7 – 9.6 were quantified by Qubit (Invitrogen) and diluted to concentration 90 ng/µl. RT master mix contained 1 µl of 1:50,000 of ERCC RNA Spike-In Mix 1 (Life Technologies) dilution for technical normalization. Altogether 169 TAC-seq detector pairs targeting biomarkers and ERCC spike-in molecules were previously mixed together from 100 µM stock solutions, creating a “100 µM” oligo pool. The mixture was diluted to 5 µM by water and stored at -20°C. 1 µl of total-RNA template for each individual library was added to 2 µl denaturation buffer, containing 5 mM Tris-HCl (pH 7.0) (Sigma-Aldrich), 1 mM dNTP mixture (Thermo Fisher), 400 nM Oligo-T30 primer and 0.05% Triton X-100 (Sigma-Aldrich). Reaction was mixed by pipetting and centrifuged briefly. RNA was denatured by 1 min at 80°C and placed on ice. After that, reverse transcriptase (RT) master mix containing 100 mM Tris-HCl (pH 8.5) (Sigma-Aldrich), 2.5 M betaine (Sigma-Aldrich), 150 mM KCl (Sigma-Aldrich), 10 mM DTT (Sigma-Aldrich), 15 mM MgCl (Sigma-Aldrich), 4 U RiboLock RNase inhibitor (Thermo Fisher) and 20 U Maxima H Minus Reverse Transcriptase (Thermo Fisher) was prepared. The content was mixed on vortex and centrifuged briefly. 2 µl of RT master mix was added to previously denatured RNA (3 µl). All RT pipetting steps were performed on ice. cDNA synthesis was performed by 30 min at 42°C, following 5 min at 85°C for RT inactivation.   Once cDNA synthesis was completed, 1 µl of 5 µM TAC-seq detector mixture was added to RT mixture. The content was mixed on vortex and centrifuged briefly. Strip tubes were placed on thermocycler, denatured 1 min at 98°C and following 1 h at 60°C to enable specific cDNA and TAC-seq probe hybridization. After hybridization, thermostable ligase reaction mixture was added on thermocycler, keeping a constant (60°C) hybridization temperature. The cycler lid was opened and strip caps were removed. 5 µl of Taq DNA ligase master mix, containing 2× Taq DNA ligation buffer (New England Biolabs, NEB) and 1 U Taq DNA ligase (NEB, cat no M0208) were added to each individual reaction tube and mixed by pipetting. The strip tubes were not removed from 60°C thermocycler to avoid self- and mispairing of TAC-seq probes. Ligation reaction was stopped after 20 min incubation by placing reaction plate on ice.   15 µl of previously combined Dynabeads MyOne Carboxylic Acid beads (2 µl) (Thermo Fisher) and 13 µl of capture buffer (30% PEG-6000 (Sigma-Aldrich), 2 M NaCl (Sigma-Aldrich), 5 mM Tris-HCl (pH 7.5) (Sigma-Aldrich), 10 mM EDTA (Sigma-Aldrich) and 0.02% Tween-20 (Sigma-Aldrich)) was added to ice-cooled ligated sample. The content was mixed by vortex. The capture was carried out for 10 min at RT, and after that the samples were placed on DynaMag-96 Side Magnet (Thermo Fisher) holding 8-well strip tubes on VersiPlate Frame (Thermo Fisher). The clean supernatant was removed. The beads on magnet were washed once with 50 µl fresh 80% ethanol. Ethanol was removed by pipetting, and the clean pellet, without ethanol drops, was dried for 2 min. Once beads were dry, strip tubes were removed from the magnet and 18 µl of PCR master mix was added directly to beads. The master mix contained 1× proofreading HOT FIREPol Blend Master Mix (SolisBiodyne, Tartu, Estonia) and 250 nM TAC-seq Left primer. In addition to universal TAC-seq Left, different TAC-seq barcode oligonucleotides were used to introduce a six-nucleotide barcode to each studied sample. For that, 2 µl of 5 µM TAC-seq Barcoded primers were added individually to each PCR reaction. Strip tubes were closed with clean domed caps, mixed on vortex until all beads were suspended. The ERCC spike-in reaction was incubated at 95°C for 12 min, followed by two cycles of 95°C for 20 s, 57°C for 60 s and 72°C for 20 s. In addition, 10 cycles of 95°C for 20 s, 65°C for 20 s and 72°C for 20 s with a final extension at 72°C for 1 min using the default ramp speed of the T100 cycler (Bio-Rad) were performed. PCR products were pooled together into 1.5 ml tube. The pooled tube was placed on magnet to remove carboxylated beads before the following column purification. Clear supernatant was purified with DNA Clean & Concentrator-5 column (Zymo Research) and eluted with 50 µl EB. The library was size-selected using AMPure XP beads (Beckman Coulter) in a single-step selection to reduce 81 bp linear PCR double-stranded by-product. 50 µl beads were added to 50 µl of the purified PCR product, incubated for 5 min at room temperature and captured by a magnet for 3 min. After incubation on magnet, the supernatant was discarded and the remaining beads were centrifuged at 500×g for 10 s. After centrifugation, the beads were placed again on the magnet and all remaining supernatant was removed. The beads were eluted directly without ethanol washing in 25 µl of EB and incubated for 1 min at RT. Finally, the eluted library was transferred to a clean tube after 1 min incubation on the magnet. The library (single 180 bp product) was visualized on a TapeStation High Sensitivity D1000 ScreenTape (Agilent Technologies) and quantified using KAPA Library Quantification Kit (Roche).  S1 TGAACT  S2 GATCTG  S3 ACATCG  S4 GCCTAA  S5 CGTGAT  S6 TGGTCA  S7 ACATGT  S8 ATTGGC  S9 CTGATC  S13 GTAGCC  S49 ATTCAG  S12 TCAAGT  S50 TACAAC  S14 TACAAG  S15 ATCAGT  S16 GCTCAT  S17 AGGAAT  S51 AACGAC  S52 GATTCC  S20 TGAGTG  S21 CGCCTG  S53 AAGCGA  S23 TGTTGG  S24 AGCATC  S25 GGTTGT  S26 CATTGT  S27 ACTCGT  S28 TATCGT  S29 CTTAGT  S30 TACTTG  S31 ATCGTG  S32 TCTCTG  S33 GTACTG  S34 ATGATG  S35 TCCATG  S36 TCGTTC  S37 GTTGTC  S38 AATGTC  S39 TATAGC  S40 CATTAC  S41 CAGGTA  S42 AGACTA  S43 CTCATA  S44 TGTAGA  S45 CTGTCA  S46 CTATCG  S47 GTAACG  S48 CGATAG Illumina NextSeq 500 high output 75 cycles kit (Illumina, San Diego, CA) using 62 bp read-1 and 6 bp index reads.