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*[[Hosuk:Lab_Notes|LabNote]] ====Day 2==== =====Image processing tools===== *Really powerful and necessary tools, expensive though *'''Imaris''' **for rendering, image processing *'''Huygens''' **for deconvolution and image alinging =====Image processing procedure===== **Take raw images data to storage server. Raw images from Confocal is 12 bit **Open raw images with Imaris, and export to Huygens **Deconvolution with Huygens, end export back to Imaris (become 32 bit) **Check images with Imaris, and reduce image size ***Maximum projection --> 2D image from 3D z stack images ***Downsampling to 8 bit ***Reduce resolution from 4000 x 4000 to 1000 x 1000 **Link time series **Create mask for aligning with Imaris and align images with Huygens **Convert time to z --> Get one image has pixel towers (stacks) of each time series of each individual rolony signal **Use genome libraries to match each colors of rolonies to known libraries, without concerning further image processing to remove noise, background and resolve which pixel is real or not. =====Rolony - Day 2===== *Cell preparation **100% confluence, and could wait one more day, and split **When cells are transferred from 6 well plate **500uL of Trypsin, incubate 5min **Add 500uL media, and mix well, and **100uL media in 96 well, or 300~350uL media in glass bottom dish **Add 60uL of cell in 96 well or 200uL in glass bottom dish **Culture one or two more day *BS(PEG)9 **20uL in 1mL PBS **Add 200uL of BS(PEG)9 mix per one glass bottome dish **Add 50uL per one well of 96 well plate *CircLigase II **Use 2uL of enzyme, but '''make 200uL mix by doubling all reagents''' **128uL H2O **20uL 10x Buffer **10uL MnCl2 **40uL Betaine **2uL CircLigase II *After CircLigase reaction (for 2hr.) **Use PBS for washing 3 times *RCA **1~2hr. for RT or 2hr. for CircLigase II would be enough, but **incubate over 12 hour *Cy3 probe hybridization (using Jonathan's sample) **1 uL of 100um probe in 1 mL 2x SSC **and add 200uL in glass bottom dish, 50uL for 96 well **Add Cy3 probe, incubate at RT for 15min **Wash once with 2x SSC **Wash once with 1x SSC **Wash with PBS, store in PBS
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