今天給大家分享一篇文獻(xiàn) ，文獻(xiàn)的名稱叫做A spatially resolved brain region- and cell typespecific isoform atlas of the postnatal mouse brain,文獻(xiàn)的地址在這里,這篇文獻(xiàn)于2021年發(fā)表于NC摘昌，其實(shí)的精髓在于運(yùn)用了三種單細(xì)胞技術(shù)，10X單細(xì)胞高蜂、10X空間轉(zhuǎn)錄組和長片段分析聪黎，值得我們借鑒一下，我們今天的任務(wù)就是來分享一下這篇文獻(xiàn)备恤。intensive reading~~

（1）簡介

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear.（腦區(qū)的可變剪切信息unclear）稿饰，We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7（對(duì)海馬和前額葉皮層 進(jìn)行 single-cell long-read sequencing研究），Isoform tests
for DIE show better performance than exon tests.（研究效果比外顯子好）露泊，We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms（檢測(cè)到數(shù)百種可溯源到細(xì)胞類型的DIE事件喉镰，這些事件通常對(duì)應(yīng)于功能不同的蛋白質(zhì)同工型 ），Mostly,one cell type is responsible for brain-region specific DIE.（一種細(xì)胞類型responsible for大腦具體區(qū)域的IDE事件）滤淳，However, for fewer genes, multiple cell types influence DIE.（較少的基因梧喷，多種細(xì)胞類型影響DIE）因此，區(qū)域識(shí)別可以（盡管很少）override細(xì)胞類型的特異性脖咐。 Cell types indigenous to one anatomic structure display distinctive DIE（一種解剖結(jié)構(gòu)固有的細(xì)胞類型顯示出獨(dú)特的DIE ）铺敌，e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage.（有點(diǎn)生澀，哈哈）屁擅，Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map（聯(lián)合分析）偿凭。Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity（意義）。
看來文章主要是研究可變剪切的空間區(qū)域性派歌，以及細(xì)胞特異性

（2）Introduction

Alternative splicing (AS) affects almost all spliced genes in mammals弯囊，vastly expands the proteome and increases functional diversity of cell types. Alternative transcription start sites (TSS) and poly-adenylation (polyA) sites further expand the alternative isoform landscape, regulating development,
differentiation, and disease痰哨。（可變剪切的作用），These RNA variables often depend on each other匾嘱，and how their combined status impacts individual molecules can only be assessed using longread sequencing（為什么做全長轉(zhuǎn)錄組斤斧，說白了就是獲得完整的可變剪切信息），which sequences transcripts in single reads with no assembly required, thereby reducing alternative transcript assembly errors and enabling accurate isoform quantification.（全長的要求）霎烙。
Brain AS is especially diverse（多樣性） and brain-region specific expression patterns of splicing factors（剪接因子） and other RNAbinding proteins drive brain-region-specific splicing. Examples
include diseases implicated by genes such as MAPT, Bin1,and neurexins（例子撬讽，可以不管他）。Brain-region-specific isoform expression can either originate from molecular regulation in one or multiple
cell types, or can arise purely from gene-expression or celltype abundance differences without splicing regulation.（調(diào)控與否）悬垃，These distinct models are especially important during postnatal development.下面的例子我們漢語翻一下游昼，在海馬和前額葉皮層，多種細(xì)胞類型經(jīng)歷了分化尝蠕，分化過程受發(fā)育特異性剪接的影響烘豌，不同于成熟細(xì)胞類型的剪接。However, no celltype-specific isoform investigation across brain regions exists to-date, owing to limitations in technology, throughput, and testing methods. HIPP and PFC are highly specialized regions of the telencephalon, and their circuitry is heavily implicated in movement control, cognition, learning, and memory formation. （有點(diǎn)專業(yè)英語的意思）看彼。Disorders involving HIPP and PFC manifest in cognitive deficits, and understanding changes occurring at crucial developmental
timepoints of these structures is important for case–control studies.Here, we employ single-cell isoform RNA sequencing (ScISOrSeq) with increased throughput in HIPP and PFC at mouse postnatal day 7 (P7) to test and define celltype-specific contributions to brain-region-specific splicing廊佩，F(xiàn)urthermore, we devised a spatial isoform expression method,which provides a spatial exon expression map in addition to the existing spatial gene expression map of the Allen developing brain atlas。（多組學(xué)分析）靖榕。
ScISOrSeq這個(gè)技術(shù)作者也是第一次聽說罐寨，當(dāng)然，諾禾單細(xì)胞現(xiàn)在推出了三代全場(chǎng)的測(cè)序服務(wù)序矩，我們往下看看鸯绿，看看結(jié)果與方法。

（3）Results

1簸淀、Short read clustering of P7 hippocampus and prefrontal cortex tissue assigns precursors to known adult cell-types.

Our ScISOrSeq approach used barcoded single cells followed by both short and long-read analyses to reveal splice variants specific to cell types.

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從技術(shù)上看瓶蝴，Barcode標(biāo)記了之后，兩種測(cè)序策略（長短兩種）租幕。短序列的測(cè)序結(jié)果用于識(shí)別細(xì)胞類型舷手，Short-read clustering across two hippocampal replicates revealed no need for integration anchors to correct for batch effects（沒有批次，牛）劲绪。然后識(shí)別細(xì)胞類型男窟，F(xiàn)urthermore, we observed six vascular and immune populations including vascular endothelial cells, microglia, and macrophages。

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2扇住、A gene-wise test to determine differential isoform expression (DIE).

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3春缕、Differential isoform expression across brain regions is governed

predominantly by one specific cell type.

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4、Cell types endogenous to one brain region have distinct splicing signatures.

5艘蹋、Choroid plexus epithelial cells (CPEs) generate distinct isoforms predominantly through alternative TSS.

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6锄贼、Single-cell basis of DIE between cell types.

When DIE is
observed between two cell types, two competing hypotheses can explain this phenomenon4. Either all cells of each cell-type behave uniformly and reflect the differences in isoform expression between the two cell types, or individual cells of one or both cell types could show variability in isoform expression. Neuronatin
(Nnat) is an important developmental gene expressing a neuronspecific isoform. In Nnat, DIE between ependymal cells and excitatory neurons is represented by the vast majority of individual cells. However, the case of DIE between excitatory neurons and granule neuroblasts is different: some granule neuroblasts
behave like excitatory neurons, while others behave like nonneurons. This may be due to different sub-populations of granule neuroblasts。

7女阀、Clustering on long-read data recapitulates short-read cell-type assignments

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我們這里主要關(guān)注幾個(gè)方法：

（1）短序列的分析（cellranger和Seurat）

The 10x cellranger pipeline (version 3.0.0) was run on the raw Illumina sequencing data to obtain single-cell expression matrices. For replicate 1, the raw expression matrices obtained through cellranger were used along with the DropletUtils package (v1.6.1) to acquire ‘eligible’ barcoded single cells (FDR < = 0.001) with UMI counts that fell below cellranger’s filtering cutoff. These barcodes were incorporated into new matrices for importing into Seurat (v 3.1). For both hippocampal replicates and the first PFC replicate, cells that had unique gene counts over 5000 or less than 700, and greater than 20% mitochondrial gene expression were removed from further analysis. To adjust for the lower mean reads/cell for the second PFC
replicate, the cutoff for minimum number of genes per cell was lowered to 350. Filtering on these parameters yielded 14,433 single cells for the hippocampus across two replicates, and 10,944 single cells for the PFC. We then used Seurat’s “merge” feature to combine the replicates for each brain region. The number of UMIs, percentage of mitochondrial gene expression were regressed from each cell and then the gene expression matrix was log normalized and scaled to 10,000 reads per cell. Next, we clustered all the cells using 30 principal components (PCs) using the Louvain algorithm with a 0.6 resolution.

（2）空間短數(shù)據(jù)的分析

The 10X spaceranger pipeline was run on raw Illumina sequencing data to obtain spatial expression matrices. Seurat’s spatial analysis functions were used to obtain gene expression similarity clusters and identify barcodes corresponding to various brain regions咱娶。

（3）Integrated analysis with published data to identify cell-types.

Published RNASeq P30 mouse brain data from Allen Brain Atlas30 was used as a reference to identify cell identities of clusters based on shared gene expression patterns. Since the Allen institute data was generated using the SmartSeq2 protocol, Seurat’s integrated anchor feature77 was used to align the two datasets and transfer cell-type labels

（4）單細(xì)胞空間聯(lián)合分析

P7 HIPP single-cell data was used as a reference to transfer labels onto P8 spatial transcriptomics data in the barcoded region corresponding to the hippocampus using Seurat’s integrated anchor feature using default parameters.

其他方法：有點(diǎn)超出本人的知識(shí)范圍。但非常重要

這里大家一起努力吧强品，革命尚未完成，我們需要學(xué)習(xí)