title: GSM6206846: Exosc3_EpiLC_CTRL_isoseq_Rep2; Mus musculus; RNA-Seq
library_construction_protocol: For RNA extraction, cells were washed 3x with DPBS and lysed directly on the plate with 1mL of TRIzol reagent (Invitrogen, 15596026). Subsequently, RNA was purified using Direct-zol RNA Miniprep Plus Kits (Zymo, R2072) following manufacturer instructions. In brief, lysed cells in TRIzol were mixed 1:1 with 100% ethanol. Mixed lysates were added to the Zymo-spin columns, treated with DNase I, and washed with provided buffers. RNA was eluted with UltraPure DNase/RNase-Free Distilled Water (Invitrogen, 10977015). For complementary DNA synthesis, 100-200 µg of DNase I treated RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). Random hexamers were used to capture total RNA. Purified RNA was submitted to the Icahn School of Medicine Genomic Core facility for sequencing. Sequencing libraries were prepared using SMARTer PCR cDNA Synthesis Kit (Clontech, 634926) per manufacturer recommendations. OligodT primers were used to capture full-length polyadenylated transcripts. cDNA was then size selected and sorted into two bins of greater or less than 4kb. These bins were pooled together at equimolar concentrations using SMRTbell Template Preparation Kit v1.0. End repaired and purified libraries were loaded onto a SMRTcell 1M, which was then sequenced on a Sequel I system with a 10-hour movie.