title: GSM6673870: Engineered Heart Tissue, treated with A61603, replicate 6; Homo sapiens; RNA-Seq
library_construction_protocol: EHTs were preserved individually with RNA stabilization solution (RNAlaterTM, ThermoFisher), stored at -80C for approx. 2 wk, and shipped on dry ice to UC Davis for RNA-seq analysis. For RNA extraction, individual EHTs were transferred to a 1.5 mL Eppendorf tube on dry ice and homogenized using a cold pestle followed by addition of 0.25 mL of Trizol (Life Technologies, 15596026), vortexed for 1 min and incubated at room temperature (RT) for 10 mins. Samples were transferred to a QIAshredder tube (Qiagen LLC, 79654), centrifuged at 4°C, 14,000g for 2 mins. Eluate was returned to the original 1.5 mL Eppendorf tube, an additional 0.75 mL of Trizol was added, vortexed for 1 min and incubated at RT for 10 mins. Chloroform (0.2 mL) was added, briefly vortexed and centrifuged at 4°C, 14,500g for 15 mins. The clear aqueous phase was transferred to a new 1.5 mL Eppendorf tube, avoiding the phenol-chloroform phase. RNA was precipitated from aqueous phase by adding 0.6 mL of isopropyl alcohol, incubating for 5 min at RT, and centrifuging at 4°C, 14,500g for 15 mins. The supernatant was carefully discarded without disturbing the pellet. Three ethanol washes were performed using 0.75 mL of 70% ETOH preceding RNA clean up. After the last wash, the tubes were left to air dry for 30 mins at RT. RNA was dissolved in 50 µL of nuclease-free water and incubated at 55°C for 5 mins. Purification of RNA was followed immediately after extraction using an RNA Clean & Concentrator kit (Zymo Research, R1017). Briefly, two volumes of RNA Binding Buffer were added to the eluate and mixed, followed by an equal amount of 100% of ETOH, mixed, and transferred to a Zymo-spin column. Sample was centrifuged at 13,000g for 30 seconds and supernatant discarded. DNase treatment was performed in column by adding 75 µL of DNA digestion buffer and 5 µL of DNase I (Zymo Research, E1010) and incubating for 15 mins at RT. Following incubation, 0.4 mL Prep Buffer was added, centrifuged at RT, 13,000g for 30 sec and supernatant discarded; 0.7 mL of RNA wash buffer was then added, centrifuged at RT, 13,000g for 30 sec and supernatant discarded. A second round of 0.4 mL of RNA wash buffer was added, centrifuged at RT, 13,000g for 30 sec and supernatant discarded. RNA was eluted by adding 30 µL of RNase-free water, incubating for 1 min, and centrifuging at RT, 13,000g for 30 seconds. Samples required an RNA integrity number (RIN) >7.0 and an A260/280 ratio of absorbance > 1.7 to proceed. Strand-specific and barcode indexed RNA-seq libraries were generated from 100-200 ng total RNA from each EHT and after poly-A enrichment using the Kapa mRNA-seq Hyper kit (Kapa Biosystems, Cape Town, South Africa) following the manufacturer instructions. The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA). The libraries were quantified by fluorometry on a Qubit fluorometer (LifeTechnologies, Carlsbad, CA) and pooled in equimolar ratios. The pool was quantified by qPCR with a Kapa Library Quant kit (Kapa Biosystems) and sequenced on an Illumina NovaSeq 6000 system (Illumina, San Diego, CA) run with paired-end 150 bp reads.