HEK3/LINC01509 Library Preparation

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.

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

Purify amplicons by 1X SPRI bead cleanup, eluting in 10µL of water.

Digest zero-length recordings with 5µL BccI in 50 uL volume:

37°C 1h 0m 0s

65°C 0h 20m 0s

On ice

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.

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

Purify amplicons by 1X SPRI bead cleanup, eluting in 10µL of water.

Take 1µL of each indexed amplicon to D1000 Tapescreen.

Pool indexed amplicons according to desired relative molarities for sequencing.