title: GSM9119479: Perturb seq in Primed cells, gRNA library, replicate1; Homo sapiens; RNA-Seq
library_construction_protocol: Chromium Next GEM Single Cell 3' HT Reagent Kits v3.1 (Dual Index) kit (10x Chromium) was used for Single Cell 3' Gene Expression and CRISPR Screening analysis. It was done by partitioning ten thousand cells per reaction into Gel Beads-in-emulsion (GEMs) The cells (16.5K cells were used in order to get recovery target of 10K cells), were then subjected to the Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10× Genomics, CA, USA) and DNA libraries protocol for generating Single Cell 3' Gene Expression and CRISPR Screening dual index libraries from single cells were prepared according to manufacture recommendations (10x Chromium). In brief, and for Single-cell RNA-seq (scRNA-seq) Gene Expression Library preparation, the pellet was cleaned up after cDNA PCR product was selected with 0.6X SPRIselect reagent (Beckman Coulter), washed twice with 80% ethanol and then eluted in 40 ul Elution buffer (EB). While for the and CRISPR Screening, 75 of the supernatants from the previous step was transferred to a new tube and 30 ul of SPRIselect reagent (Beckman Coulter) 1.2X was added and then washed twice with 80% ethanol and finally eluted in 40 ul EB. Both libraries were prepared for sequencing following manufacturer company instructions (10x Chromium, CG000316, Chromium Next GEM Single Cell 3ʹ Kit v3.1, 16 rxns PN-1000268 ). Sequencing was done using Illumina Novaseq6000 platform with following sequencing conditions: 28 bp (Read1) and 90 bp (Read2). For the primed perurb-seq, 2 samples were sequenced in 2 batches. For the naïve experiment, 2 samples were sequenced in the same batch.