GSM7775286: Perturb-seq Amplicon library; Mus musculus; OTHER
A single cell suspension was loaded into the 10X Chromium device and libraries were prepared using the 10X Single Cell 3' Library & Gel Bead Kit v3.1 (10X Genomics, PN-1000121), following manufacturer's instructions. Amplicon sgRNA PCRs were performed for each of the 24 full length 10X cDNA samples of SAM ESCs transduced with the 475 sgRNA library, as previously described (Hill et al., 2018). Brieﬂy, 10 ng of full length 10X cDNA were used as starting material and each round of PCR ampliﬁcation with the primers described in Hill et al., 2018 (see also Table S6) was monitored by KAPA SYBR (KR0389) to avoid overcycling. After multiplexing, these enrichment sgRNA libraries were sequenced across two lanes of the Illumina Hiseq2500 Rapid Run, with 27 cycles for read 1, 267 cycles for read 2 and 8 cycles for i7 sample index.Indexed sequencing libraries were constructed using the reagents in the GemCode Single-Cell 30 Library Kit, following these steps: (1) end repair and A-tailing; (2) adapter ligation; (3) postligation cleanup with SPRIselect; (4) sample index PCR and cleanup. The barcode sequencing libraries were quantiﬁed by quantitative PCR (KAPA Biosystems Library Quantiﬁcation Kit for Illumina