OKSM reprogramming intermediates from Mouse Embryonic Fibroblasts
strain
Black6-129X1/SvJ
cell type
OKSM reprogramming intermediates from MEFs
age
E13.5 embryos
time
day 3
antibody
none (input)
genotype/variation
Col1a1::tetOP-OKSM ; R26-M2rtTA
protocol
Renilla control knockdown
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
To generate ATAC-seq libraries, total of 50000 cells were used and libraries were constructed as previously described46. Briefly, cells were washed in PBS twice, counted and nuclei were isolated from 100000 cells using 100ul hypotonic buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40) to generated two independent transposition reactions. Nuclei were split in half and treated with 2.5 μL Tn5 Transposase (Illumina) for 30min at 37°C. DNA from transposed nuclei was then isolated and PCR amplified using barcoded Nextera primers (illumina). For all ChIP experiments 10e7 reprogramming intermediates were collected per library. Chromatin precipitation assays were performed as previously described69 using goat polyclonal Anti-Sox2 (AF2018, R&D). Briefly, cells were cross-linked on plate in 1% methanol free formaldehyde and snap frozen in liquid nitrogen until processed. Nuclei were isolated using 1ml of Cell lysis buffer (20mM Tris pH8, 85mM KCL, 0.5% NP40 and 1X HALT protease inhibitor cocktail) and resuspended in nuclear lysis buffer (10mM Tris-HCL pH7.5, 1% NP40, 0.5% Na deoxycholate, 0.1% SDS, 1X HALT protease inhibitor cocktail) and sonicated using optimized pulses of branson sonifier (1min ON/OFF pulses for 5 cycles) for ChIP-seq libraries and S220 Covaris sonicator (Settings: duty cycle 5%, intensity 6, cycles/burst 200, pulse length 60s, 20 cycles, 8°C) for sono-seq input preparations. Sonications were verified for both methods using the 2100 Bioanalyzer. Immunoprecipitations were carried out by first adjusting salt concentration in sheared chromatin to 167mM NaCL and adding antibodies (6ug of Sox2 antibody) and incubated for 3-4 hrs at 4C. 50μl Protein G Dynabeads (Invitrogen) were prepared for each IP reaction by washing 2-3 times in chip dilution buffer (16.7mM Tris-HCl pH 8.1, 167mM NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA) and added for an additional hour to pull down bound chromatin. Beads complexes were then washed 6 times in RIPA buffer (20mM Tris-HCL pH8.1, 1mM EDTA, 140mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Na deoxyhcholate), then two times with RIPA with high salt concentration (500mM), then 2 washes in LiCL buffer (10mM tris-HCL pH8.1, 1mM EDTA, 1% DOC, 1% NP40, 250mM LiCL) and two final washes in TE. Complexes were then eluted and reverse cross-linked in 50ul ChIP elution buffer (10mM Tris-HCL pH8, 5mM EDTA, 300mM NaCl, 01% SDS) and 8ul of reverse crosslinking buffer (250mM Tris-HCl pH 6.5, 1.25M NaCl, 62.5mM EDTA, 5mg/mL Proteinase K, 62.5ug/mL RNAse A by incubation at 65°C for 6h. DNA was isolated using Ampure SPRI beads and yield quantified using Qubit fluorometer. Libraries for sequencing were prepared using standard Illumina protocols.