Conditional requirement for dimerization of the membrane-binding module for BTK signaling in lymphocyte cell lines

Bruton’s tyrosine kinase (BTK) is a major drug target in immune cells. The membrane-binding pleckstrin homology and tec homology (PH-TH) domains of BTK are required for signaling. Dimerization of the PH-TH module strongly stimulates the kinase activity of BTK in vitro. Here, we investigated whether BTK dimerizes in cells using the PH-TH module and whether this dimerization is necessary for signaling. To address this question, we developed high-throughput mutagenesis assays for BTK function in Ramos B cells and Jurkat T cells. We measured the fitness costs for thousands of point mutations in the PH-TH module and kinase domain to assess whether dimerization of the PH-TH module and BTK kinase activity were necessary for function. In Ramos cells, we found that neither PH-TH dimerization nor kinase activity was required for BTK signaling. Instead, in Ramos cells, BTK signaling was enhanced by PH-TH module mutations that increased membrane adsorption, even at the cost of reduced PH-TH dimeriz..., RNA-seq libraries from BTK-transduced and CD69 selected or input cells were prepared from TRI reagent-extracted samples as follows, beginning with the reverse-transcription. Following precipitation, RNA was resuspended in water containing 5 µM RT primer. The mixture was heated to 65ºC for 5 min, then snap cooled on ice. The RNA–oligo mixture was then mixed with 1x first-strand buffer (Thermo Fisher), 0.5 mM deoxynucleotide triphosphates, 10 mM DTT, 1 µL SuperaseIn (Thermo Fisher), and 0.5 µL Superscript III (Thermo Fisher). The final reaction mixture was incubated at 50ºC for 1 h. Followed this incubation, RNA was hydrolyzed by addition of 5 µL of 1 M NaOH and incubation at 90ºC for 10 min. The mixture was then neutralized with 25 µL of 1 M HEPES, pH 7.4, and desalted using a Micro Bio-Spin P-30 column, in tris (BioRad), eluting the cDNA in 60 µL. cDNA was amplified using PCR, which also attached the Miseq hybridizing sequences. Fastq files from MiSeq runs were aligned to the Fasta file..., , # Differential requirement for dimerization of the membrane-binding PH-TH module of BTK in B cells and T cells

[https://doi.org/10.5061/dryad.sbcc2frd9](https://doi.org/10.5061/dryad.sbcc2frd9)

The counts of each variant in each library are uploaded here as tab-delimited text files. **Fasta files** that were used to generate these counts are also uploaded here. 

## Description of the data and file structure

There are 106 data files deposited here. 6 of these files (with suffix \"fasta\") are the files that contain the nucleotide sequence of all BTK variants used in this study.

Note: Jurkat T cells and Ramos B cells, which are human cancer cell lines.

The remaining 100 files (with suffix \"_P.tsv\", for \"processed\") contain the read counts for each sequence in each experiment. Each of these files has four fields in the file name, separated by an underscore.

The naming scheme of these files are as follows:

(1) The first field is the cell line from which it was derived (\"Jurkat\" or \"Ra...,