GSM5666304: MDAMB231 cells with knockout of USP2 at 96h replicate 2 [sample_522]; Homo sapiens; RNA-Seq
Cells were lysed 96 hours post crRNA transfection or 24 hours post inhibitor treatment; the plates were washed one time with PBS on a plate washer (BioTek). The PBS was removed (leaving ~15 uL/well residual volume), and 30 uL/well 1 X lysis buffer (1x Qiagen TCL, 1% beta-mercaptoethanol) was added. The plates were incubated for 5 minutes at room temperature to aid cell lysis, and then frozen at -80˚C until RNA extraction. The DGE RNA-seq was performed as previously published (Semrau et al., 2017; Soumillon et al., 2014) with modifications described previously (Schauer et al., 2020) (full protocol at https://www.protocols.io/view/3-39-dge-high-throughput-rna-library-preparation-bumynu7w). The Nextera DNA kit was used to prepare the sequencing library following the manufacturer's instructions. 55 ng of cDNA was tagmented for 5 minutes at 55 ̊C and purified using a Zymo DNA Clean & Concetrator-5 column. The cDNA was amplified (7 cycles) then purified using a 0.9x ratio of AMPure XP magnetic beads. The Agilent BioAnalyzer HS DNA Kit was used to assess the library size distribution before qPCR quantification and sequencing at the Harvard Medical School Biopolymers Facility (paired end sequencing was performed on an Illumina NextSeq). high-throughput, low-cost RNA-sequencing method (3' Digital Gene Expression or 3'DGE-seq)