Table of contents

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Efficient generation of functional CFTR-expressing airway epithelial cells from human pluripotent stem cells pp363 - 381

Amy P Wong, Stephanie Chin, Sunny Xia, Jodi Garner, Christine E Bear & Janet Rossant


Functional CFTR-expressing airway epithelial cells are generated via differentiation firstly into progenitors and then into mature epithelia with apical CFTR activity.

Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy pp382 - 396

Huayan Yang, Shouning Yang, Jilie Kong, Aichun Dong & Shaoning Yu


This protocol describes how in-solution protein FTIR can be used to obtain information about the relative contributions of α-helices, β-sheets, β-turn, and random coil structures to a protein's secondary structure.

An optogenetics- and imaging-assisted simultaneous multiple patch-clamp recording system for decoding complex neural circuits pp397 - 412

Guangfu Wang, Daniel R Wyskiel, Weiguo Yang, Yiqing Wang, Lana C Milbern, Txomin Lalanne, Xiaolong Jiang, Ying Shen, Qian-Quan Sun & J Julius Zhu


This protocol describes how to achieve optogenetics- and imaging-aided physiological analysis of synaptic interconnections among 4–30 simultaneously and sequentially recorded neurons.

The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells pp413 - 425

Sarah X L Huang, Michael D Green, Ana Toste de Carvalho, Melanie Mumau, Ya-Wen Chen, Sunita L D'Souza & Hans-Willem Snoeck


This protocol describes how to direct differentiation of human pluripotent stem cells into developmental lung progenitors, and subsequently into predominantly distal lung epithelial cells, by following four stages that recapitulate lung development.

Building high-quality assay libraries for targeted analysis of SWATH MS data pp426 - 441

Olga T Schubert, Ludovic C Gillet, Ben C Collins, Pedro Navarro, George Rosenberger, Witold E Wolski, Henry Lam, Dario Amodei, Parag Mallick, Brendan MacLean & Ruedi Aebersold


Targeted proteomics by SRM/MRM or SWATH MS relies on reference assay libraries for peptide identification. This protocol is for building extensive, high-quality reference libraries starting with data acquired by discovery proteomics.

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues pp442 - 458

Je Hyuk Lee, Evan R Daugharthy, Jonathan Scheiman, Reza Kalhor, Thomas C Ferrante, Richard Terry, Brian M Turczyk, Joyce L Yang, Ho Suk Lee, John Aach, Kun Zhang & George M Church


Lee et al. provide their protocol for fluorescent in situ sequencing (FISSEQ) of RNA. This technique allows spatial and quantitative information about mRNA expression to be obtained simultaneously.

3D computational reconstruction of tissues with hollow spherical morphologies using single-cell gene expression data pp459 - 474

Robert Durruthy-Durruthy, Assaf Gottlieb & Stefan Heller


In this protocol, the authors describe an approach for PCA-based 3D reconstruction of hollow sphere–shaped tissues and organs from single-cell gene expression data.

MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing pp475 - 483

Mark A Urich, Joseph R Nery, Ryan Lister, Robert J Schmitz & Joseph R Ecker


This protocol describes how to prepare a sequencing library for MethylC-seq analysis, enabling the methylation status of cytosines in genomic DNA to be determined at nucleotide resolution on a genome-wide scale.

Supercooling preservation and transplantation of the rat liver pp484 - 494

Bote G Bruinsma, Tim A Berendsen, Maria-Louisa Izamis, Heidi Yeh, Martin L Yarmush & Korkut Uygun


With this protocol rat livers can be kept viable for up to 96 h and transplanted successfully. The livers are first loaded with cryoprotectants to prevent ice formation and protect against hypothermic injury, and they are then cooled to –6 °C without freezing.

Surgical models of Roux-en-Y gastric bypass surgery and sleeve gastrectomy in rats and mice pp495 - 507

Bote G Bruinsma, Korkut Uygun, Martin L Yarmush & Nima Saeidi


This protocol describes mouse and rat models of Roux-en-Y gastric bypass and sleeve gastrectomy, which are similar to bariatric operations performed to treat obese humans.

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems pp508 - 516

Martin D Witte, Tongfei Wu, Carla P Guimaraes, Christopher S Theile, Annet E M Blom, Jessica R Ingram, Zeyang Li, Lenka Kundrat, Shalom D Goldberg & Hidde L Ploegh


Sortase A can be used for the site-specific modification of proteins. This protocol describes its immobilization on Sepharose beads, allowing for larger-scale continuous flow reactions or easy separation from the enzyme in batch reactions.

Statistical analysis of cell migration in 3D using the anisotropic persistent random walk model pp517 - 527

Pei-Hsun Wu, Anjil Giri & Denis Wirtz


This protocol enables the statistical analysis of cell motility in 2D and 3D microenvironments using the anisotropic persistent random walk model, providing a new tool to characterize 3D cell migration.

Production of an acetone-butanol-ethanol mixture from Clostridium acetobutylicum and its conversion to high-value biofuels pp528 - 537

Sanil Sreekumar, Zachary C Baer, Anbarasan Pazhamalai, Gorkem Gunbas, Adam Grippo, Harvey W Blanch, Douglas S Clark & F Dean Toste


There is a growing demand for renewable transportation fuels. This protocol describesthe production of an acetone-butanol-ethanol mixture in Clostridiumacetobutylicum as well as its reaction to form long-chain ketones for testing as car or jet fuel.


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