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== Fluidigm C1 Run: Human Pyramidal Nuclei == *Samples prepared and sorted by Yun at Scripps (FACS ARIA) *Bulk sorted nuclei (BD Influx) based on PI positive - ~120 nuclei/ul *Centrifuged nuclei at 2000rpm 4C for 10 min (table-top centrifuge) *Resuspended in low volume, added PI stain again to visualize nuclei and counted - ~250 nuclei/ul *Used ERCC diluted 1:20,000 (10 fold less than previous experiment) *Add 15ul of nuclei to 10ul cell suspension reagent and loaded 20ul onto chip (5ul actually loading into chip = '''750 nuclei''') *Observed chip under microscope and counted occupancy rate: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Capture Site''' | align="center" style="background:#f0f0f0;"|'''# of Cells''' | align="center" style="background:#f0f0f0;"|'''Capture Site''' | align="center" style="background:#f0f0f0;"|'''# of Cells''' |- | 1||0||49||0 |- | 2||1||50||0 |- | 3||3||51||0 |- | 4||0||52||0 |- | 5||0||53||0 |- | 6||0||54||1 |- | 7||2||55||0 |- | 8||1||56||1 |- | 9||0||57||0 |- | 10||0||58||0 |- | 11||0||59||0 |- | 12||0||60||1 |- | 13||0||61||1 |- | 14||1 frag||62||1 |- | 15||0||63||0 |- | 16||1||64||0 |- | 17||1||65||0 |- | 18||0||66||1 |- | 19||1 + 1 frag||67||0 |- | 20||0||68||2 |- | 21||1||69||0 |- | 22||6||70||0 |- | 23||1||71||0 |- | 24||0||72||0 |- | 25||1||73||0 |- | 26||0||74||0 |- | 27||0||75||0 |- | 28||3||76||1 |- | 29||1||77||0 |- | 30||0||78||0 |- | 31||0||79||0 |- | 32||0||80||0 |- | 33||0||81||2 |- | 34||1 + 1 frag||82||1 |- | 35||0||83||0 |- | 36||0||84||0 |- | 37||3||85||0 |- | 38||0||86||0 |- | 39||1||87||0 |- | 40||0||88||0 |- | 41||1||89||1 |- | 42||0||90||0 |- | 43||0||91||0 |- | 44||4||92||0 |- | 45||0||93||1 |- | 46||1||94||0 |- | 47||3||95||0 |- | 48||6||96||0 |- | |} '''Plate Layout:''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' | align="center" style="background:#f0f0f0;"|'''5''' | align="center" style="background:#f0f0f0;"|'''6''' | align="center" style="background:#f0f0f0;"|'''7''' | align="center" style="background:#f0f0f0;"|'''8''' | align="center" style="background:#f0f0f0;"|'''9''' | align="center" style="background:#f0f0f0;"|'''10''' | align="center" style="background:#f0f0f0;"|'''11''' | align="center" style="background:#f0f0f0;"|'''12''' |- | A||3_C03||1_C02||0_C01||0_C49||0_C50||0_C51||0_C06||0_C05||0_C04||0_C52||0_C53||1_C54 |- | |||||||||||||||||||||||| |- | B||0_C09||1_C08||2_C07||0_C55||1_C56||0_C57||0_C12||0_C11||0_C10||0_C58||0_C59||1_C60 |- | |||||||||||||||||||||||| |- | C||0_C15||1f_C14||0_C13||1_C61||1_C62||0_C63||0_C18||1_C17||1_C16||0_C64||0_C65||1_C66 |- | |||||||||||||||||||||||| |- | D||1_C21||0_C20||1 + 1f_C19||0_C67||2_C68||0_C69||0_C24||1_C23||6_C22||0_C70||0_C71||0_C72 |- | |||||||||||||||||||||||| |- | E||1_C25||0_C26||0_C27||0_C75||0_C74||0_C73||3_C28||1_C29||0_C30||0_C78||0_C77||1_C76 |- | |||||||||||||||||||||||| |- | F||0_C31||0_C32||0_C33||2_C81||0_C80||0_C79||1 + 1f_C34||0_C35||0_C36||0_C84||0_C83||1_C82 |- | |||||||||||||||||||||||| |- | G||3_C37||0_C38||1_C39||0_C87||0_C86||0_C85||0_C40||1_C41||0_C42||0_C90||1_C89||0_C88 |- | |||||||||||||||||||||||| |- | H||0_C43||4_C44||0_C45||1_C93||0_C92||0_C91||1_C46||3_C47||6_C48||0_C96||0_C95||0_C94 |- | |||||||||||||||||||||||| |- | |} Note: f=fragmented nuclei == RNA Extraction of Unfixed and Fixed Nuclei == Samples: #Bulk unfixed and sorted human nuclei #Bulk fixed and sorted Nuclei #Bulk fixed and unsorted nuclei #300 fixed and sorted nuclei #300 fixed and sorted nuclei #150 fixed and sorted nuclei #150 fixed and sorted nuclei #30 fixed and sorted nuclei #30 fixed and sorted nuclei *RNA extraction using the ZR RNA Microprep kit (with on column DNase treatment) and collected in 6ul *Determined concentration for bulk samples using Qubit RNA kit - only detected RNA for the unfixed nuclei - fixation (1%paraformaldehyde) seemed to inhibit RNA extraction
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