Implementation of methods for toxicological profiling of engineered nanomaterials (ENMs) using transcriptomics, proteomics and metabolomics
Report on RNA Preparation  Total RNA from treated or untreated cells were extracted by TRIzol® following the manufacturer’s protocol (illustrated in Figure 3). Briefly, 1 ml of TRIzol® reagent (Invitrogen) was added to cells per well of a 6-well plate (or up to 10 million cells). The cells in TRIzol® were homogenized by passing through a 26 G needle 8-10 times and incubated at room temperature for 5 min. After homogenization, 0.2 ml of chloroform was added per mL TRIzol® lysate and mixed by vigorous shaking for 15 sec. The mixture was incubated at room temperature for 3 min, transferred to a pre-spun 2ml Phase-Lock-Gel tube (5 Prime), and centrifuged at 18,000 x g for 15 min at 4°C to separate phase. The upper aqueous phase containing RNA was carefully aspirated to a fresh, nuclease-free tube. The RNA was then precipitated by adding one volume of isopropanol and 1/10 volume of 5 M NaCl, and centrifuged at 18,000 x g for 10 min at 4°C. Finally, the RNA pellet was washed with 75% ethanol, air-dried and dissolved in nuclease-free water. The dissolved total RNA was quantified by NanoDrop (NanoDrop Technologies) and the quality was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies) according to the manufacturer's instructions.      Report on Setup for RNA-seq  Stranded total RNA sequencing was performed using 500 ng total RNA following the TruSeq stranded total RNA library preparation protocol (Illumina) as illustrated in Figure 4. Briefly, ribosomal RNA depleted total RNA was obtained using the RiboZero Gold depletion system from the TruSeq stranded total RNA kit. Purified total RNA was fragmented and the fragmented RNA was purified by Agencourt RNAClean XP SPRI beads (Agencourt, A63987) and converted to first strand cDNA using random hexmer primer (Invitrogen) and Supescript II (Invitrogen), followed by second strand cDNA synthesis with E.coli DNA pol I (Invitrogen) and RNAse H (Invitrogen) and Agencourt AMPure XP beads (Agencourt) purification. Then the paired-end sequencing library was prepared from purified double stranded (ds) cDNA. In short, the ds cDNA was end repaired and ligated with Illumina paired end barcoded sequencing adapters (Illumina). Each step was purified by Agencourt AMPure XP SPRI beads (Agencourt). The purified ligated product was amplified by Phusion polymerase with the following PCR program: 98°C 30 sec, (98°C, 10 sec, 65°C 30 sec, 72°C 30 sec) x 15 cycles, 72°C 5 min, 4°C hold. The PCR product was then purified by Agencourt AMPure XP beads (Agencourt) as the sequencing library. The prepared library was quantified by Qubit using DNA HS kit (Invotrogen), quality assessed by Bioanalyzer, and sequenced on Illumina HiSeq for 2 x 100 cycles following the manufacturer’s instructions.