title: GSM8756588: CLBL-1, w12, p121, Ibr+AS, 72 h; Canis lupus familiaris; RNA-Seq
library_construction_protocol: A total volume of 6 mL medium containing 2 x 106 cells was seeded into each well of 6-well plates, and then exposed to 1 μM Ibrutinib, 5 μM AS-605240, their combination, or DMSO, two wells per treatment. When reaching 24, 48, and 72 hours respectively, incubated cells were harvested and washed three times with cool PBS and centrifuged at 200x g for 8 min at 4° C. QIAzol® Lysis Reagent (QIAGEN, Venlo, Netherlands) of 700 μL was added into the pellet of 5 x 106 cells to lyse. Total RNA was isolated using the miRNeasy Mini Kit (QIAGEN, Venlo, Netherlands) referring to the manufacturer's Quick-Start Protocol. Genomic DNA contamination was prevented through the RNase-free DNase (QIAGEN, Venlo, Netherlands) digestion on the RNeasy® Mini column. Finally, the concentration and quality of RNA in each sample was evaluated by Nanodrop Spectrophotometer ND1000 (PEQLAB Biotechnologie GmbH, Erlangen, Germany). A total RNA of 2 μg (with RNA integrity number > 8) was used to prepare the sequencing libraries. Referring to the manufacturer's instruction, poly-A RNA was enriched and ligated to sequencing adapters with the NEBNext Ultra II RNA Library Prep Kit (New England Biolabs, Ipswich, USA). Single-end sequencing was conducted on Illumina NextSeq500 sequencing platform (Illumina, San Diego, USA) to generate 75 bp-long reads.