title: GSM4506041: Time_course_day4; Mus musculus; RNA-Seq
library_construction_protocol: Skin ILCs were isolated from ears of IL-5 Fate IL-17AGFP and IL-22BFP mouse reporters from naïve mice (day 0) and at daily intervals thereafter along a four-day time course of IL-23 treated mice (1,2,3, and 4 days post injection; n=5 mice per group). Total skin ILCs were sorted as CD45.2, CD90.2 positive, and lineage, TCRgamma and CD3epsilon negative. Sorted cells were washed with PBS/0.04% BSA and processed for droplet-based 3′ end massively parallel scRNA-seq: sorted ILCs were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 3′ Reagent Kits V3 according to the manufacturer's protocol (10X Genomics). scRNA-seq libraries were sequenced using a 75 cycle Nextseq 500 High Output kit.   To quantify Cre, as a proxy for Il5 expression, a Cre-specific primer was selected ~200bp upstream from the polyA signal motif (AAUAAA) for the Cre that was used to generate the mouse in this study. Primers were ordered from Genewiz. Cre primer 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGTGTCCATCCCTGAAAT -3', P7-index primer: 5'- CAAGCAGAAGACGGCATACGAGAT[index]GTCTCGTGGGCTCGGAGATGTG -3', P5 primer: 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC -3'  PCR was conducted with 20μl reaction consisting of 2μl dilute template, 2μl of 10μM P5 primer, 2μl of 1μM of Cre-primer, 2μl of 10μM P7 index primer, 2μl H2O and 10μl of Q5® High-Fidelity 2X Master Mix (New England Biolabs) using the following PCR program:: 98°C for 30sec, 22 cycles of 98°C for 10seq, 68°C for 30sec, and 72°C for 30sec followed by 72°C for 50sec and hold at 12°C. Libraries were then pooled equally by volume, purified by MinElute (Qiagen), and eluted in 35μl double distilled water. Purified libraries were loaded on Blue Pippin for size selection of 400-520nt. Tapestation D1000 was used for quality control after size selection. Qubit HS DNA was used to quantify the library. The library was sequenced on an Illumina MiSeq with a V3 150 cycle kit with the following setting: Read1: 28; Read2: 125; I7 Index: 8; I5 Index: 0  YFP was amplified from 10x WTA mixture with P5 primer and Yfp-primer, followed by Sanger Sequencing with P5 and YPF primer. The Sanger sequencing result was then compared to the vector sequence used in the generation of the mice.  Yfp-primer: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCGTGACCACCTTCGG 10x RNAseq (3', V3)
attributes.tag: GEO Accession
attributes.value: GSM4506041