AGO2 immunoprecipitation RNA-seq in T-ALL cell lines
AGO2 immunoprecipitation RNA-seq in T-ALL cell lines
For each immunoprecipitation reaction, 3 × 10^7 cells were used. 10% of input (3 x 10^6) was collected as total RNA (TOT) fraction. Cells were cultured under standard conditions in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 10% (JURKAT, LOUCY, DND-41) or 20% (ALL-SIL) of fetal bovine serum (Thermo Fisher Scientific). AGO2 immunoprecipitation (AGO2-IP) was performed as described previously with some minor adaptations [doi: 10.1093/nar/gkp715]. Briefly, cell lysates were incubated with EZview Red Protein G Affinity Gel (Sigma-Aldrich, St. Louis, MO, USA) coated with anti-AGO2 antibody (Clone 2E12-1C9, Abnova, Taipei, Taiwan) at 4°C overnight. Anti-IgG antibody was used as a negative control (Merck, Darmstadt, Germany). 10% of input, AGO2-IP and flow-through fractions were collected to test the effectiveness of the AGO2 RNA IP procedure via AGO2 immunoblotting and RT-qPCR for selected miRNAs. The remaining AGO2-IP fraction was further used for RNA isolation and sequencing. The miRNeasy micro RNA isolation kit (Qiagen, Hilden, Germany) was used for the extraction of total RNA (TOT). RNA isolates were DNase-treated and purified with the use of RNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA). AGO2-IP RNA was isolated directly using the RNA Clean and Concentrator Kit. RNA concentration was measured with Quantus Fluorometer (Promega, Madison, WI, USA) using Qubit HS RNA Assay Kit (Thermo Fisher Scientific). RNA integrity was determined with 4200 Tapestation using High Sensitivity RNA ScreenTape (Agilent Technologies, Santa Clara, CA, USA).   Libraries were constructed from the total (TOT) and AGO2-IP RNA fraction samples in the 4 T-ALL cell lines, using TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat protocol, based on ribo-depletion. Fragmentation was performed at 90′C for 2 minutes, as described previously [https://doi.org/10.1016/j.ygeno.2021.10.018].