HEK3/LINC01509 Library Preparation
Mathew Chu
Abstract
Library preparation for HEK3 recording site (LINC01509) using PCR and digestion.
Steps
HEK293T Cell Lysis
Make up 1:50 Thermolabile Proteinase K (NEB) in Viagen (cell).
Resuspend cells in 25µL of Viagen/Proteinase K.
Quickly transfer cells into a PCR strip to avoid viscosity.
Perform lysis in a PCR machine:
37°C 1h 0m 0s
55°C 0h 30m 0s
On ice
Store lysates at -20°C in the pre-PCR freezer.
PCR Amplification of Genomic DNA
Make up a 100µL PCR reaction per sample (Q5) using 10µL of cell lysate.
98°C 0h 0m 30s
98°C 0h 0m 5s
68°C 0h 0m 10s
72°C 0h 0m 20s
72°C 0h 2m 0s
SPRI Bead Concentration
Purify amplicons by 1X SPRI bead cleanup, eluting in 10µL of water.
Digestion of Zero-Length Recordings
Digest zero-length recordings with 5µL BccI in 50 uL volume:
37°C 1h 0m 0s
65°C 0h 20m 0s
On ice
Gel Separation and Extraction of Recordings
Perform gel electrophoresis in 4% LMP agarose at 3 V/cm for 1h 0m 0s.
Extract the region from 300 bp to 500 bp and elute in 12µL of water.
Store gel products at -20°C in the post-PCR freezer.
Indexing PCR of Recombinant DNA
Using 1µL of gel product, perform indexing PCR in 12µL volume with Nextera primers .
98°C 0h 0m 30s
98°C 0h 0m 5s
67°C 0h 0m 10s
72°C 0h 0m 20s
72°C 0h 2m 0s
SPRI Bead Purification
Purify amplicons by 1X SPRI bead cleanup, eluting in 10µL of water.
D1000 Tapescreen
Take 1µL of each indexed amplicon to D1000 Tapescreen.
Pool indexed amplicons according to desired relative molarities for sequencing.
