title: GSM8682452: In_vivo_GF, sub5, RNA, Rep2; Mus musculus; OTHER
library_construction_protocol: RNA was harvested using Rneasy mini kit (Qiagen) from HepG2 or Rneasy midi kit (Qiagen) from mouse liver. DNA was extracted using mini-prep kit (Qiagen) Plasmid DNA library preparation: we used 200 ng DNA as a template and each library was assigned unique i5 and i7 primers to aid in demultiplexing post-sequencing. The library was PCR amplified using 2X KAPA HiFi HotStart ReadyMix (Roche) with the following program (98°C for 3min; 98°C for 15s, 65°C for 30s, 7°2C for 45s and repeat for 17 cycles); 72°C for 2min). PCR reactions from each sample were combined and purified using 0.8X KAPA HyperPure Beads (Roche). RNA library preparation: we first isolated mRNA from 40 ug of total RNA from each sample using Dynabead Oligo-dT25 beads (Invitrogen) for HepG2 samples. We used 600 ug of total RNA to isolate mRNA for mouse liver samples. RNA was treated with Turbo DNase (Thermo Fisher Scientific) to remove contaminating DNA, and reverse-transcribed with SuperScript III (Thermo Fisher Scientific) using gene-specific RT primers. The library was PCR amplified using 2X KAPA HiFi HotStart ReadyMix (Roche) and 10 uM P7_UMI_Primer with the following program (98°C for 60s; 65°C for 30s; 72°C for 45s). Samples were purified using 1.4X KAPA HyperPure Beads (Roche) and eluted with 20uL water at 37°C for 3 min at 300 rpm on the thermomixer. Purified samples from the earlier step were used as a template per PCR reaction. The library was PCR amplified using 2X KAPA HiFi HotStart ReadyMix (Roche), 10 uM junction_fwd primer, and 10 uM P7_junction_rev primer with the following program (98°C for 90s; 98°C for 15s, 65°C for 30s, 72°C for 40s and repeat for 22 cycles; 72°C for 2min). Samples were purified using 0.8X KAPA HyperPure Beads (Roche) and eluted with 20uL water at 37°C for 3 min at 300 rpm on the thermomixer. Lastly, each library was assigned a unique i5 primer to aid in demultiplexing post-sequencing The library was PCR amplified using 2X KAPA HiFi HotStart ReadyMix (Roche) and 10uM P7_seqready rev primers with the following program (98°C for 90s; 98°C for 15s, 65°C for 30s, 72°C for 40s and repeat for 8 cycles; 72°C for 2min). PCR reactions from each sample were combined and purified using 0.8X KAPA HyperPure Beads (Roche). Samples were eluted with an appropriate amount of water at 37°C for 3 min at 300 rpm on the thermomixer. Sample concentration post-purification was measured using Qubit™ dsDNA Quantification Assay Kits (Invitrogen).