title: GSM8284894: TCells_4T1_In_vivo_Cas9_rep1; Mus musculus; RNA-Seq
library_construction_protocol: FACS-purified cells were lysed immediately upon direct sorting in 150 µL of extraction buffer from the Arcturus PicoPure RNA isolation kit (Thermo Scientific). The quality of the RNA isolated through the PicoPure kit was determined with a TapeStation RNA High sensitivity Screen Tape (Agilent). Due to the low concentration of total RNA the SMARTer® Stranded Total RNA-Seq Kit v3 -Pico Input Mammalian (Takara Bio) was used to prepare sequencing libraries. Briefly, total RNA samples (0.27–8 ng) were mechanically fragmented (4 minutes) and reverse-transcribed using random priming into double-stranded cDNA in the presence of a template switch oligo (TSO). When the reverse transcriptase reaches the 5' end of the RNA fragment, the enzyme's terminal transferase activity adds non-templated nucleotides to the 3' end of the cDNA, then the TSO pairs with the added non-templated nucleotide, enabling the reverse transcriptase to continue replicating to the end of the oligonucleotide. This procedure results in a cDNA fragment that contains sequences derived from the random priming oligo and the TSO. So, PCR amplification using primers binding to the latter sequences was performed so to add full-length Illumina adapters, including dual barcodes for multiplexing. Ribosomal cDNA was cleaved by ZapR in the presence of the mammalian-specific R-Probes. Remaining fragments were enriched with a second round of PCR amplification using primers designed to match Illumina adapters. Since RNA input concentrations was highly variable the samples were grouped into two sets of samples and PCR cycle numbers were adjusted (Group 1 = 6.5-8ng input, 12 cycles; Group 2 = 0.27-4 ng, 16 cycles). The quality and quantity of the enriched libraries were validated using a TapeStation analyzer (Agilent). The product presented as a smear with an average fragment size of approximately 300 - 360 bp. The libraries were normalized to 5nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.