SPT16_AID_V5_Clone2_3-IAA_Rep3 [TT-seq]
source_name
SPT16_AID_V5_Clone2_3-IAA
cell line/type
E14 mES cell line
tag integration
osTIR1 was integrated into the genome using CRISPR-directed homologous recombination; separately, Supt16 was C-terminally tagged with the 37 kDa mini-AID tag and a triple V5 tag
treatment
Cells were left undisturbed on 10 cm plates for 48 hours prior to treatment. Cells were treated with 500 nM 3-IAA mixed into medium for 24 hours. Cells were incubated at 37 degrees Celsius with 5% CO2 for the duration of treatment.
rna extraction
Cells were trypsinized, quenched, pelleted, washed with cold PBS, and crosslinked with 1% formaldehyde for 15 minutes. Crosslinking was quenched with .5M glycine for 5 minutes. Fixed cells were pelleted and washed with cold PBS
molecule subtype
4sU-labeled RNA