GSM8605829: EGFP 3 week Mouse liver #2; Mus musculus; RNA-Seq
2 weeks and 3 weeks later, mice were perfused, and liver tissues were isolated. The whole median lobe was collected. Three 3-4mm punches from the Median Lobe were collected and placed in separate cryotubes, snap frozen in liquid nitrogen, and stored in -80°C. Remains from the median lobe were placed in a cryotube, snap frozen in liquid nitrogen, and stored in -80°C. To prepare viral DNA for long-read sequencing,100 uL of rAAV material was digested in proteinase K reaction at 55°C for 30 minutes followed by deactivation of proteinase K at 80°C for 15 minutes, and gradually dropping the temperature to 4°C to allow single stranded DNA to anneal to double stranded DNA. After this, DNA was purified by Monarch DNA and PCR Purification Kit (NEB, Cat: #T1130) following the manufacturing protocol starting mixture of one volume of sample buffer and three volumes of binding buffer.  DNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and sample purity and integrity was checked using Nanodrop and Agilent TapeStation. Following quality control, PacBio SMRTbell amplicon libraries for PacBio Sequel were constructed using SMRTbell prep kit 3.0 (PacBio, Menlo Park, CA, USA) using the manufacturer recommended protocol. Primer annealing, polymerase binding, and complex cleanup was performed using the Sequel binding kit 3.1 & cleanup beads (PacBio) and loaded onto PacBio Sequel IIe. Sequencing was performed on PacBio Sequel IIe SMRT cell with AAV Mode enabled. Sequel IIe sequencing generated subread files, as well as HiFi reads via on-board circular consensus sequencing (CCS) analysis.RNA was extracted from fresh frozen tissue samples using Qiagen RNeasy Plus Universal mini kit following manufacturer's instructions (Qiagen, Cat: 74881). RNA samples were quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). ERCC Ex-fold RNA reagent (Cat: 4456739) from ThermoFisher Scientific, was added to normalized total RNA prior to library preparation following manufacturer's protocol. Samples were treated with TURBO DNase (Thermo Fisher Scientific, Cat: AM2238) to remove DNA contaminants. The next steps included performing rRNA depletion using QIAseq® FastSelect™−rRNA HMR kit (Qiagen, Cat: 334386), which was conducted following the manufacturer's protocol. RNA sequencing libraries were constructed with the NEBNext Ultra II RNA Library Preparation Kit for Illumina (NEB, Cat: E7770) by following the manufacturer's recommendations. Briefly, enriched RNAs are fragmented for 15 minutes at 94 °C. First strand and second strand cDNA are subsequently synthesized. cDNA fragments are end repaired and adenylated at 3'ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).