Sf 900 2 sfm medium

E. coli cells were cultured in LB medium (Sigma) at 37 °C. The E. coli strain Stellar cells were used to generate and amplify plasmids for SPNS2 mutations and strain DH10Bac cells were used to generate bacmids.
Sf9 insect cells (Thermo Fisher Scientific) were cultured in Sf-900 II SFM medium (GIBCO) at 27 °C. The baculovirus were generated and amplified in Sf9 cells in Sf-900 II SFM medium supplemented with 2% (v/v) fetal bovine serum (FBS) (GIBCO).
Expi293F suspension cells were cultured in Freestyle 293 expression medium (GIBCO) and grown in an orbital shaker at 37 °C and 8% CO₂.
HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO) supplemented with 10% FBS and grown in an incubator at 37 °C and 5% CO₂.

Pab1 and PABPC1 full-length and truncation constructs were heterologously expressed in the E. coli expression strain Rosetta DE3. Not1(754-1000) was heterologously expressed in the E. coli expression strain BL21 DE3. Cells were grown to an OD600nm of 1.0 at 37°C in standard TB medium, the temperature reduced to 18°C and expression induced by the addition of IPTG at a final concentration of 250 μM. Cells were harvested 16 h post induction. Both yeast and human recombinant Pan2-Pan3 complexes as well as the core yeast Ccr4-Caf1 nucleases were expressed in the Trichoplusia ni Hi5 cell line. All Pan2-Pan3 complexes were subcloned in the pFL expression vector and co-expressed in insect cells using the Multibac method (Fitzgerald et al., 2006 (link)). The Ccr4(110-837)-Caf1(146-433) complex was expressed from the pFastBac DUAL vector (Invitrogen). Cells were transfected with recombinant Baculovirus variants carrying Pan2-Pan3 or Ccr4(110-837)-Caf1(146-433) coding sequences and grown for 48 to 60 h in Sf-900 II SFM Medium (Thermo Fisher Scientific) at 27°C. Recombinant Baculovirus generations were amplified in the Spodoptera frugiperda Sf21 cell line grown in Sf-900 II SFM Medium at 27°C.