title: GSM9159647: pLN cKO_2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated using NucleoSpin RNA (Takara Ref: 740955.50) and the quality and quantity were measured using Tapestation 4200 (Agilent Technologies) and Qubit (Invitrogen) respectively. Takara SMART-Seq mRNA Kit was used to generate full-length cDNA from 10ng total RNA samples by following the manufacturer's instructions. The process started with first-strand cDNA synthesis, where the 3' SMART-Seq CDS Primer II A and the SMART-Seq v4 Oligonucleotide were used to prime and template switch at the 5' end of the transcript. This step was followed by LD PCR amplification using the PCR Primer II A to amplify the cDNA. The amplified cDNA was then purified using the Agencourt AMPure XP Kit to remove any contaminants. Finally, the quality and quantity of the cDNA were validated using Tapestation 4200 (Agilent Technologies) and Qubit (Invitrogen) respectively. The full length cDNA fragments were converted into libraries using Nextera XT DNA library kit (Catalog No.: FC-131-1096). 150pg was used for tagmentation using Nextera transposon, in which the full length cDNA were fragmented and tagged with the adapter sequences. These fragments were further amplified using indexed primers resulting in Illumina compatible libraries. The quality and quantity were measured using Tapestation 4200 (Agilent Technologies) and Qubit (Invitrogen) respectively. Prior to sequencing, libraries were diluted to 4nmoles and pooled.