title: GSM4190258: MPs_colon_1_day_12_1.12.14; Mus musculus; RNA-Seq
library_construction_protocol: Femurs and tibias of donor mice were removed and BM was flushed with cold PBS. BM was washed with cold PBS twice and filtered by 100 μm filter. Cells were suspended with PBS and loaded on equal amount of Ficoll (GE healthcare). Tubes were centrifuged 920 g in room temperature for 20 minutes without breaks and Buffy coats were collected and washed with cold PBS. CD11c-DTR > wt]. Cells were stained and sorted according to the following markers: CD117- CD11b+ CD115+ Ly6C+ GFPint. Intestines were removed and fecal content flushed out with PBS; tubes were opened longitudinally and cut into 0.5 cm sections. Pieces were placed in 5ml / sample (up to 300gr of tissue) of Hanks' Balanced Salt Solution (HBSS) with 10% heat-inactivated FCS/FBS, 2.5mM EDTA and 1mM DL-Dithiothreitol ((DTT), Sigma-Aldrich Cat# D9779 ) and incubated on a 37oC shaker at 300rpm for 30 minutes to remove mucus and epithelial cells. Following incubation, samples were vortexed for 10 seconds and filtered through a crude cell strainer. Pieces that did not pass the strainer were collected and transferred to 5ml/sample of PBS +/+ with 5% heat-inactivated FCS/FBS, 1 mg/ml Collagenase VIII (Sigma-Aldrich Cat# C2139 ) and 0.1 mg/ml DNase I (Roche Cat# 10104159001 ). Tissue was incubated in a 37oC shaker at 300 rpm for 40 minutes (colon) or 20 minutes (ileum) in the solution. After incubation, samples were vortexed for 30 seconds until tissue was dissolved, then filtered through a crude cell strainer. The strainer was washed with PBS -/- and centrifuged in 4oC, 375G for 6 minutes. Cells were stained and subjected to FACS analysis or sorting. Cells were sorted into 100 µl of lysis/binding buffer (Life Technologies) and stored at 80 °C. mRNA was captured using Dynabeads oligo(dT) (Life Technologies) according to manufacturer's guidelines. A derivation of MARS-seq 21 was used  to prepare libraries for RNA-seq, as detailed in Shemer et al. 45. RNA-seq libraries were sequenced using the Illumina NextSeq 500. Raw reads were mapped to the genome (NCBI37/mm9) using hisat (version 0.1.6). Only reads with unique mapping were considered for further analysis. Gene expression levels were calculated and normalized using the HOMER software package (analyzeRepeats.pl rna mm9 -d<tagDir>-count exons -condenseGenes -strand + -raw). Gene expression matrix was clustered using k-means algorithm (MATLAB function kmeans) with correlation as the distance metric. PCA was performed by MATLAB function pca. Gene ontology was performed by DAVID (https://david.ncifcrf.gov). Data on molecules and pathways was partly obtained by Ingenuity Pathway Analysis (IPA), Ingenuity Target Explorer, Qiagen and Metascape Pathway analysis 23.
attributes.tag: GEO Accession
attributes.value: GSM4190258