RNA-seq of ReNcell VM treated with 3 uM torin1
A multiwell cell dispenser (catalog# 5840300, Thermo Scientific, Waltham, MA) with standard tubing (catalog# 24072670, Thermo Scientific, Waltham, MA) was used to plate 2500 neural stem cells (ReNcell VM, catalog# SCC008, Millipore, Billerica, MA) into each well of a 384-well cell culture plate (Perkin Elmer, Waltham, MA). Neural stem cells where differentiated into mature neural cells for one week and then treated with compounds or DMSO using a D300 Digital Dispenser (Hewlett-Packard, Palo Alto, CA). D300 software was used to randomize dispensation of compounds. After 24 hours, the cells were washed once with PBS using an EL405x plate washer (BioTek, Winooski, VT) leaving 5-10 ul of PBS behind in each well. 10 ul of 1X TCL lysis buffer (catalog# 1070498, Qiagen, Hilden, Germany) with 1% (v/v) mercaptoethanol was added per well, and the plates were stored at -80Â°C until the RNA extraction was performed. For RNA extraction, the cell lysate plate was thawed and centrifuged for 1 min at 1000 rpm. Using a BRAVO (Agilent, Santa Clara, CA) liquid handler, the lysate was mixed thoroughly before transferring 10 ul to a 384 well PCR plate. 28 ul of home-made Serapure SPRI beads (GE Healthcare Life Sciences, Marlborough, MA)were added directly to the lysate, mixed and incubated for 5 min. The plate was transferred to a magnetic rack and incubated for 5 min prior to removing the liquid to aggregate the beads. The beads were washed with 80% ethanol twice, allowed to dry for 1 min, 20 ul of nuclease-free water was added per well, the plate was removed from the magnetic rack and the beads were thoroughly resuspended. Following a 5 min incubation, the plate was returned to the magnetic rack and incubated an additional 5 min before transferring the supernatant to a fresh PCR plate. 5 ul of the RNA was transferred to a separate plate containing RT master mix and 3' and 5' adapters for reverse transcription and template switching, and incubated for 90 min at 42Â°C. The cDNA was pooled and purified with a QIAquick PCR purification kit according to the manufacturers directions with the final elution in 21 ul of nuclease-free water. This was followed by exonuclease I treatment for 30 min at 37Â°C that was stopped with a 20 min incubation at 80Â°C. The cDNA was then amplified using the Advantage 2 PCR Enzyme System (Takara, Fremont, CA) for 6 cycles, and purified using AMPure XP magnetic beads (Beckman Coulter Genomics, Chaska, MN).Library preparation was performed using a Nextera XT DNA kit (Illumina, San Diego, CA) on 5 reactions per sample following the manufacturers instructions, amplified 12 cycles, and purified with AMPure XP magnetic beads (Beckman Coulter Genomics, Chaska, MN). The sample was then quantified by qPCR and sequenced on a single Illumina NextSeqrun with 75bp paired-end reads at the Harvard University Bauer Core Facility. The raw fastq files were processed using the bcbio-nextgen single cell/DGE RNA-seqanalysis pipeline (https://bcbio-nextgen.readthedocs.io/). Before submission, the fastq file was demultiplexed into separate files using the sample-specific barcodes in the first read and unique molecular identifiers were used to filter duplicate reads of the same original mRNA molecule.