Clarity otx lysis loading buffer v2

HEL cells were
counted using a Countess Automated Cell Counter (Invitrogen) or Countess
3 Automated Cell Counter (Invitrogen) and seeded in 24-well plates
(Techno Plastic Products 92024) at 115,000 live cells per mL and 500
μL per well in complete medium. After 3–4 h, treatments
were performed by diluting oligonucleotides or conjugates in 50 μL
of Opti-MEM (Gibco 31985047) and directly adding the diluted oligonucleotides
or conjugates to the cells. After 24 h, the cells were transferred
to clean tubes and pelleted by centrifugation. The cells were washed
with ice-cold phosphate-buffered saline (PBS; Gibco 10010015), ice-cold
0.1 mg/mL heparin (Sigma-Aldrich H3149) in PBS, and then with ice-cold
PBS. The cells were lysed in Clarity OTX Lysis-Loading Buffer v2.0
(Phenomenex AL0-8579). Cell lysates were centrifuged at 13,000 rpm
and 4 °C for 10 min, and supernatants were transferred to clean
tubes prior to storage at −80 °C. Lysates were thawed
on ice and diluted 1:75 in ultrapure water prior to analysis by CL-qPCR.

Subcellular
fractions were prepared using the Rapid, Efficient, And Practical
(REAP) method^{48 (link)} with minor changes. Briefly,
HEL cells were counted, seeded, and treated as described above. After
24 h, the cells were transferred to clean tubes and pelleted by centrifugation.
The cells were washed with ice-cold PBS (Gibco 10010015), ice-cold
0.1 mg/mL heparin (Sigma-Aldrich H3149) in PBS, and then with ice-cold
PBS. Cellular membranes were lysed with ice-cold 0.1% (v/v) Nonidet
P 40 (Fluka 74385) in PBS. Samples were centrifuged at 1000g and 4 °C for 1 min, and supernatants were transferred
to clean tubes prior to storage at −80 °C (cytoplasmic
fractions). Nuclear pellets were washed with ice-cold 0.1% (v/v) Nonidet
P 40, followed by ice-cold PBS. The nuclei were lysed in Clarity OTX
Lysis-Loading Buffer v2.0 (Phenomenex AL0-8579). Nuclear lysates were
centrifuged at 13,000 rpm and 4 °C for 10 min, and supernatants
were transferred to clean tubes prior to storage at −80 °C
(nuclear fractions). All fractions were thawed on ice. Cytoplasmic
fractions were diluted 1:1 in Clarity OTX Lysis-Loading Buffer v2.0.
Then, diluted cytoplasmic fractions and nuclear fractions were diluted
1:75 in ultrapure water prior to analysis by CL-qPCR.