John Blair
@jblairsci.bsky.social
Postdoc in the Satija Lab at NYGC. Former PhD at Bateup Lab at UC Berkeley
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John Blair
@jblairsci.bsky.social
· Oct 23
This paper was entirely led by Izzy and I was an accessory in the wet lab performing the necessary validation experiments alongside others - its exciting stuff!
John Blair
@jblairsci.bsky.social
· Oct 23
She observed that the knocking down RPL10 and RPL24 has the same effect as knocking down RPL5 and RPL11, known members of the ribosomal stress pathway, showing that her fingerprinting-identified candidates were indeed real!
John Blair
@jblairsci.bsky.social
· Oct 23
Next, she identified two proteins that were likely involved in the ribosomal stress response from their transcriptome profile. We used FlexPlex to knock down those genes with siRNA, treated them with Nutlin-3a, oligohashed them and then used custom probes to confirm the loss of the intended target.
John Blair
@jblairsci.bsky.social
· Oct 23
From there she determined that only under certain doses were these inhibitors HDAC-specific and above a certain threshold, they then act like pan-HDAC inhibitors
John Blair
@jblairsci.bsky.social
· Oct 23
First, she was curious about the specificity of HDAC inhibitors that are purported to be HDAC subclass specific versus pan-HDAC inhibitors, so we applied FlexPlex with oligo-based hashing to simultaneously observe the effect of these compounds on the transcriptome and histone modifications
John Blair
@jblairsci.bsky.social
· Oct 23
There is a lot in this paper and I won't go into the method, but will highlight how we applied FlexPlex within the manuscript to help validate some of the phenomena that Izzy discovered using RNA fingerprinting
John Blair
@jblairsci.bsky.social
· Oct 23
Mapping transcriptional responses to cellular perturbation dictionaries with RNA fingerprinting
Single-cell perturbation dictionaries provide systematic measurements of how cells respond to genetic and chemical perturbations, and create the opportunity to assign causal interpretations to observa...
www.biorxiv.org
The second paper I want to highlight was led my Izzy Grabski, where she developed a new method of "RNA Fingerprinting" to map transcriptional responses to perturbation dictionaries
www.biorxiv.org/content/10.1...
www.biorxiv.org/content/10.1...
John Blair
@jblairsci.bsky.social
· Oct 23
Overall, this paper was led by myself and carried out by a number of people including @abradu.bsky.social , Izzy Grabski, Carol Dalgarno and of course Rahul Satija.
John Blair
@jblairsci.bsky.social
· Oct 23
We also identified new candidates for involvement in the integrated stress response, including RACK1, showing an increase in ATF4 levels upon perturbation
John Blair
@jblairsci.bsky.social
· Oct 23
One observation of interest was a set of genes that seemingly responded to feedback from mTOR signaling - going up with both Ragulator and TSC loss and going down with mTORC1 and GATOR1 loss - sets of complexes that have opposite effects on mTOR signaling
John Blair
@jblairsci.bsky.social
· Oct 23
Finally, we clustered perturbations from the validation FlexPlex assay into different categories based on their transcriptome and we observed how the different perturbations converge on mTOR signaling
John Blair
@jblairsci.bsky.social
· Oct 23
We validated 120 of these hits with another FlexPlex assay, observing pRPS6 changes in most of them.
John Blair
@jblairsci.bsky.social
· Oct 23
However, we wanted to bring these predictions genome-wide - so we applied the model to the K562 GWPS dataset from the Weissman Lab, observing 586 perturbations in that dataset that we predicted to significantly alter pRPS6
John Blair
@jblairsci.bsky.social
· Oct 23
From there we modeled pRPS6 activity based on transcriptomic profile using RIDGE regression and applied it to other datasets from with known or inferred changes in pRPS6 - including datasets in completely different cell types including organoids!
John Blair
@jblairsci.bsky.social
· Oct 23
We then curated that list to 50 of the top perturbations producing higher or lower pRPS6 and ran a FlexPlex assay with that, producing a “glossary” dataset with paired transcriptome-pRPS6 profiles for each perturbation.
John Blair
@jblairsci.bsky.social
· Oct 23
We then applied FlexPlex to understand regulators of mTORC1 signaling. We first did a genome-wide bulk CRISPRi screen on pRPS6 in K562 cells to provide a candidate list of relevant perturbations which, while noisy, reconstructed the known mTOR pathway and revealed new potential regulators.
John Blair
@jblairsci.bsky.social
· Oct 23
We demonstrate the effectiveness of FlexPlex through perturbing known components of the mTOR pathway, observing increases in pRPS6 in cells with TSC1 knocked down and decreases in cells with RPTOR knocked down. We confirmed excellent guide assignment and robust transcriptome profiling with high UMIs
John Blair
@jblairsci.bsky.social
· Oct 23
Iterative, multimodal, and scalable single-cell profiling for discovery and characterization of signaling regulators
Cell signaling plays a critical role in regulating cellular state, yet uncovering regulators of signaling pathways and understanding their molecular consequences remains challenging. Here, we present ...
www.biorxiv.org
I wanted to highlight two recent papers from myself and colleagues!
In the first, we develop a method, FlexPlex, that leverages the 10x Genomics Flex kits to capture RNA, intracellular protein and guide RNAs in single cells with high resolution multiplexing
www.biorxiv.org/content/10.1...
In the first, we develop a method, FlexPlex, that leverages the 10x Genomics Flex kits to capture RNA, intracellular protein and guide RNAs in single cells with high resolution multiplexing
www.biorxiv.org/content/10.1...
John Blair
@jblairsci.bsky.social
· Mar 9
John Blair
@jblairsci.bsky.social
· Mar 6
I will make Helen and Tommy get bluesky and then i will @ them in a subsequent post, but thank you for reading!!
John Blair
@jblairsci.bsky.social
· Mar 6
This work was the last year or so of my Ph.D in the Bateup Lab as covid hit, and I didn’t have an opportunity to complete it.
Very fortunately, Tommy came along and added SO much (immunopanning, 4i, additional scRNA-seq, westerns and all the analysis) making it way better than i ever could have!
Very fortunately, Tommy came along and added SO much (immunopanning, 4i, additional scRNA-seq, westerns and all the analysis) making it way better than i ever could have!
John Blair
@jblairsci.bsky.social
· Mar 6
There is a ton of IF showing the remarkable pathology of both the knockout organoid astrocytes (left) and astrocytes from resected patient cortical tuber tissue
John Blair
@jblairsci.bsky.social
· Mar 6
The single cell sequencing was performed at multiple important timepoints in early development and in multiple genetic backgrounds, all with our meticulously engineered conditional two-hit TSC2 knockout alleles and knockout reporter cassettes
John Blair
@jblairsci.bsky.social
· Mar 6
mTORC1 activation drives astrocyte reactivity in cortical tubers and brain organoid models of TSC
Tuberous Sclerosis Complex (TSC) is a genetic neurodevelopmental disorder associated with early onset epilepsy, intellectual disability and neuropsychiatric disorders. A hallmark of the disorder is co...
www.biorxiv.org
Excited (again) to see this paper finally in (pre)print!
We did single-cell sequencing on our TSC model organoids and observed strong signatures of reactive astrogliosis (neat)- then we looked at resected patient brain tissue and saw the same thing! (More neat!)
www.biorxiv.org/content/10.1...
We did single-cell sequencing on our TSC model organoids and observed strong signatures of reactive astrogliosis (neat)- then we looked at resected patient brain tissue and saw the same thing! (More neat!)
www.biorxiv.org/content/10.1...
John Blair
@jblairsci.bsky.social
· Feb 28
Also I couldn't have done it without the fellowship support of the @jcchildsfund.bsky.social - thank you!
John Blair
@jblairsci.bsky.social
· Feb 28
Phospho-seq
A highly-customizable Hugo research group theme powered by Wowchemy website builder.
phospho-seq.com
This method is adaptable to many cell types and systems which we have tried internally - if you are interested in trying this out, please get in touch! Additionally, phospho-seq.com has more resources with protocols and data from each tested antibody, with more to come!
John Blair
@jblairsci.bsky.social
· Feb 28
There are significant changes from the initial pre-print posted .... some time ago.... including profiling retinal organoids, adding new benchmarks and demonstrating an additional method to incorporate transcriptomic data
John Blair
@jblairsci.bsky.social
· Feb 28
I had a lot of help with this paper and special thanks to my collaborators from the Treutlein Lab including @epign.bsky.social ! (see, some people ARE on here).
John Blair
@jblairsci.bsky.social
· Feb 28
Phospho-seq: integrated, multi-modal profiling of intracellular protein dynamics in single cells - Nature Communications
Here, the authors demonstrate Phospho-seq, a single-cell multiomics method capable of quantifying chromatin accessibility alongside intracellular proteins, including post-translationally modified prot...
www.nature.com
Happy that my first paper from my Postdoc is out where we perform Phospho-seq, a single-cell ATAC-sequencing with intracellular protein quantification in human brain organoids: www.nature.com/articles/s41...