title: GSM7100301: Before_3; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Valencia, CA). Amplification and barcoding of sgRNAs was performed following the manufacturer's specifications for constructing CRISPR pooled lentiviral sgRNA libraries (Cellecta, Mountain View, CA). Briefly, an initial round of 25 cycles of PCR was performed using 100ng genomic DNA to amplify sgRNA sequences.  Amplicons from the first PCR were used in a second PCR of 9 cycles to add index, P5, and P7 sequences for Illumina sequencing. The final sequencing libraries were quantified using the Qubit dsDNA assay (ThermoFisher) and assessed for size using the Tapestation D1000 assay (Agilent). The pooled sequencing libraries were then size-selected for the size range of 340-425bp using the PippinHT (Sage Science). Libraries were sequenced on a NextSeq550 High-output flowcell (Illumina) to obtain single-end 50-nt reads.