ena-STUDY-QBlab-05-12-2020-12:53:46:106-254
scRNA-seq for HeLa-AB1 and F9-AB2 cells to research the control of gene expression noise.
HeLa-AB1 and F9-AB2 cells were seeded to in ibidi 24-well µ-plate at 30,000 cells per well one day prior to experiments. At day 0, two wells (A2, A3) and one well (C1) of HeLa-AB1 cells were replenished with regular media and regular media containing 330 nM LMK-235 and 1 µM A-485, respectively, and illuminated for two days with 21 and 9 µW/cm2, respectively.  At the same time, two wells (A1, B1) and one well (C1) of F9-AB2 cells with regular media and regular media containing 1 µM LMK-235 and 1 µM A-485, respectively, and continue culture at dark for 2 days.F9-AB2 cells (three samples named A1, B1 and C1 respectively) and HeLa-AB1 cells (four samples named A2, A3 and C2, respectively) in ibidi 24-well µ-plate were washed with 500 µL DMEM for three times, following incubated in 125 µL TrypLE Express Enzyme at 37 ? for 5 minutes. This enzyme was deactivated by 250 µL DMEM+10%FBS and the cells were transferred into 1.5 mL centrifuge tubes and centrifuged down at 200 g, 4 ? for 5 min. Cell pellets were washed with 1 mL PBS with 0.1% w/v BSA. Discard the PBS as much as possible and resuspend the cell pellets with an additional 100 µL PBS containing 0.1% BSA. The cells were filtered through the filter cap of flow tube (Catalog no. 352235; BD Falcon). Aliquots of 10 µL filtered cells were mixed with equal volumes of trypan blue solution for cell counting with the Countstar analyzer. Finally, the human cells (HeLa-AB1 A2, A3, and B2) and mouse cells (F9-AB2, A1, B1 and C1) were mixed at 1:1 ratio to form three samples (A1/A2, B1/A3, C1/C2).  Subsequently, according to the 10× Genomics user guide, scRNA-seq libraries were constructed in three steps including GEM Generation & Barcoding, Post GEM-RT Cleanup & cDNA Amplification and 3' Gene Expression Library Construction. In the first step, in order to obtain a final recovery of 10,000 cells/sample, 16,500 cells/sample were loaded into the 10× machine (Chromium Next GEM Single Cell 3' Library & Gel Bead Kit v3.1). In the third step, after the scRNA-seq libraries constructed, each library is split into two fractions. The one was sequenced on Hiseq X system at HaploX Biotech, while the other was sequenced on MGI 2000 platform at BGI.
HeLa-AB1 and F9-AB2 cells were seeded to in ibidi 24-well µ-plate at 30,000 cells per well one day prior to experiments. At day 0, two wells (A2, A3) and one well (C1) of HeLa-AB1 cells were replenished with regular media and regular media containing 330 nM LMK-235 and 1 µM A-485, respectively, and illuminated for two days with 21 and 9 µW/cm2, respectively.  At the same time, two wells (A1, B1) and one well (C1) of F9-AB2 cells with regular media and regular media containing 1 µM LMK-235 and 1 µM A-485, respectively, and continue culture at dark for 2 days.F9-AB2 cells (three samples named A1, B1 and C1 respectively) and HeLa-AB1 cells (four samples named A2, A3 and C2, respectively) in ibidi 24-well µ-plate were washed with 500 µL DMEM for three times, following incubated in 125 µL TrypLE Express Enzyme at 37 ? for 5 minutes. This enzyme was deactivated by 250 µL DMEM+10%FBS and the cells were transferred into 1.5 mL centrifuge tubes and centrifuged down at 200 g, 4 ? for 5 min. Cell pellets were washed with 1 mL PBS with 0.1% w/v BSA. Discard the PBS as much as possible and resuspend the cell pellets with an additional 100 µL PBS containing 0.1% BSA. The cells were filtered through the filter cap of flow tube (Catalog no. 352235; BD Falcon). Aliquots of 10 µL filtered cells were mixed with equal volumes of trypan blue solution for cell counting with the Countstar analyzer. Finally, the human cells (HeLa-AB1 A2, A3, and B2) and mouse cells (F9-AB2, A1, B1 and C1) were mixed at 1:1 ratio to form three samples (A1/A2, B1/A3, C1/C2).  Subsequently, according to the 10× Genomics user guide, scRNA-seq libraries were constructed in three steps including GEM Generation & Barcoding, Post GEM-RT Cleanup & cDNA Amplification and 3' Gene Expression Library Construction. In the first step, in order to obtain a final recovery of 10,000 cells/sample, 16,500 cells/sample were loaded into the 10× machine (Chromium Next GEM Single Cell 3' Library & Gel Bead Kit v3.1). In the third step, after the scRNA-seq libraries constructed, each library is split into two fractions. The one was sequenced on Hiseq X system at HaploX Biotech, while the other was sequenced on MGI 2000 platform at BGI.