title: GSM4041732: Invitro_Tpt3D_IL7_CGRP_B1; Mus musculus; RNA-Seq
library_construction_protocol: Lungs were digested with the lung dissociation kit (Miltenyi Biotec) and subsequently enriched for CD90.2+ cells using CD90.2 MicroBeads (Miltenyi Biotec). Lung ILCs were isolated by fluorescence-activated cell sorting as 7AAD- CD45+ CD90.2+ CD127+ Lineage (CD3, CD4, CD8, CD11b, CD11c, CD19, NK1.1, TCRβ, TCRγδ)- cells. Two replicates from each batch of distinct experiments were sequenced. RNA was quantified using a Qubit RNA HS Assay kit (Invitrogen), and 2ng of RNA were used as input for a modified SMART-Seq2 protocol (Picelli et al., 2014) entailing RNA secondary structure denaturation (72˚C for three minutes), reverse transcription with Maxima Reverse Transcriptase (Life Technologies), and whole transcriptome amplification (WTA) with KAPA HiFi HotStart ReadyMix 2X (Kapa Biosystems) for 12 cycles. WTA products were purified with Ampure XP beads (Beckman Coulter), quantified with a Qubit dsDNA HS Assay Kit (Invitrogen), and quality accessed with a High Sensitivity DNA Chip run on a Bioanalyzer 2100 system (Agilent). 0.2 ng of purified WTA product was used as input for Nextera XT DNA Library Preparation Kit (Illumina). Uniquely barcoded libraries were pooled and sequenced with a NextSeq 500 sequencer using a high output V2 75 cycle kit (Illumina) and 2x38 paired end reads.